By Cortney Pellettieri
Good Housekeeping: What's the best part of your day?
Heidi Klum: When the kids [Leni, 6; Henry, 5; Johan, 4; and Lou, 1] say, "I love you."
GH: What object do you treasure the most?
HK: My family photo albums. I have tons. From all our beautiful "weddings" (my husband, Seal, and I do a vow renewal each year) to our children's birthday parties, all the photos are organized and bound in leather albums. It's so old-fashioned and nice. They are really special keepsakes.
GH: In your home, what is there always time for?
HK: Kisses, hugs, giggles, making a mess, 6 P.M. dinners. In other words, a full and affectionate family life.
GH: What's on your nightstand right now?
HK: Pictures of the kids, our wedding photo, and the book The White Tiger by Aravind Adiga. On my husband's side is one of the first photos we took together when we met.
GH: What's a date night for you and your husband like?
HK: Snuggling in bed and watching a movie together, because I wind up falling asleep in the kids' rooms when I put them to bed. If we do go out, I like to dress up, do my makeup, and wear something sexy for him.
GH: What would your husband say your best quality is?
HK: I'm his best friend, I listen, and I'm by his side, no matter what.
GH: And your worst?
HK: I'm not that patient sometimes. I'm like a rocket - I go a hundred miles per hour.
GH: What's one beauty product you can't live without?
HK: Does a toothbrush count? I like a clean mouth.
GH: Any parenting rules you swear by?
HK: Many, but "Listen to your parents" - that's an über-important one.
GH: Any parenting rules you break?
HK: Sometimes the kids can sit in the living room with plates on their laps to eat and watch TV, but that's very rare.
GH: Do you want to have more kids?
HK: Four is a lot of children. Now we're complete; we look around the table and feel like there's no one missing. Everyone who is supposed to be around the table is there.
GH: What's the hardest part about having four kids?
HK: Making sure you give each one the time, love, and attention he or she needs. We have to remember that even Johan, who goes through life so easily, still needs just as much attention as everyone else.
Originally published on February 8, 2011
生一场闷气：闷气一生，自然见啥摔啥，有利于加强运动，消耗脂肪。同时精神的消耗会让你产生厌倦感，自然提不起食欲，减少了食品的摄入，这样下来减一斤没问题吧。
GH提示：生闷气前，妥善安置家里的贵重物品。
看一部恐怖片：深更半夜独自一人观赏，最好中途再安排个朋友帮你拉闸断电，一惊一诧，冷汗直冒，浑身哆嗦，反复几次，出汗消耗体能排出毒素，哆嗦增加身体摆动，一场电影看下来再减一斤。
GH提示：如果担心自己承受力有限，提前穿条纸尿裤以备不测……
绝食一天：如果你够狠心、够坚定，甚至可以滴水不进。假装自己正坚守上甘岭，不但磨练了意志品格，再加上完全没有摄入，还可以逼出身体的水分，最少也减一斤吧。
GH提示：手机要随身携带，万一体力不支栽倒在马路上也好及时拨打120。
熬一个通宵：不管是泡吧，还是上网，反正整晚熬着。当你精疲力竭地仰望早上初升的太阳，不减才怪。
GH提示：牛黄清心丸、痤疮膏要准备好，因为打乱作息的后果就是内分泌混乱。
吃一顿泻药：一天跑上十几次，拉得头昏眼花脚发软，一顿不行来两顿……
GH提示：千万不要跑到街上去，最好能跟马桶保持10米范围内的距离，否则……
去工行排队交按揭：这绝对是让你焦灼不安的耐力活，你的体力在和银行办公速度以及超级少的办公窗口的搏斗中会得到最充分的消耗。
K歌：唱歌可以燃烧脂肪，若加上空肚子和载歌载舞的效果，减掉的脂肪也十分可观。以下是推荐的减肥歌曲：崔健的《一无所有》、周杰伦的《双截棍》、迪克牛仔的《忘记我还是忘记他》。
吹气球：吹气球的时候肺活量增大，对腹部的肌肉有着很好的锻炼作用。
两个浴缸洗澡：准备两个浴缸，一边45℃水，一边15℃水，轮流换着泡，据说放点绿茶渣效果更好。
贴电话卡：理论是，磁性作用会调整血和气的流动，若是贴在穴道上就更能收到效果。以后电话卡里的钱用完了，千万把卡留着，也算是资源的整合再利用。
1Cancer and Haematology Division, The Walter and Eliza Hall Institute of Medical Research (WEHI) and the Cooperative Centre for Cellular Growth Factors, Parkville, Victoria, Australia.
2Department of Molecular Medicine, Karolinska Institute, Karolinska Hospital, Stockholm, Sweden.
3Center for Bone Research at the Sahlgrenska Academy, Department of Internal Medicine, Gothenburg University, Gothenburg, Sweden.
4Amrad Corporation Ltd., Burnley, Victoria, Australia.
Abstract
Mice deficient in SOCS2 display an excessive growth phenotype characterized by a 30–50% increase in mature body size. Here we show that the SOCS2–/– phenotype is dependent upon the presence of endogenous growth hormone (GH) and that treatment with exogenous GH induced excessive growth in mice lacking both endogenous GH and SOCS2. This was reflected in terms of overall body weight, body and bone lengths, and the weight of internal organs and tissues. A heightened response to GH was also measured by examining GH-responsive genes expressed in the liver after exogenous GH administration. To further understand the link between SOCS2 and the GH-signaling cascade, we investigated the nature of these interactions using structure/function and biochemical interaction studies. Analysis of the 3 structural motifs of the SOCS2 molecule revealed that each plays a crucial role in SOCS2 function, with the conserved SOCS-box motif being essential for all inhibitory function. SOCS2 was found to bind 2 phosphorylated tyrosines on the GH receptor, and mutational analysis of these amino acids showed that both were essential for SOCS2 function. Together, the data provide clear evidence that SOCS2 is a negative regulator of GH signaling.
See the related Commentary beginning on page 233
Introduction
Growth hormone (GH) is the major growth factor of the somatotrophic axis involved in the promotion of postnatal longitudinal growth. It acts via direct and indirect mechanisms, including the use of second messengers such as IGF-1. GH also has other potent effects, including modulation of lipid, glucose, nitrogen, and mineral metabolism, promotion of lipolysis, increase of amino acid uptake and protein synthesis, and decrease of protein breakdown (1). In contrast, chronic elevations of the hormone, as seen in acromegalic patients, causes gigantism, hyperinsulinemia, impaired glucose tolerance, insulin resistance, and finally diabetes (2).
The number of clinical applications of GH are increasing, but one of the most important has been the stimulation of growth in children. Clinical studies have demonstrated that daily exogenous GH injection is a successful treatment for growth disorders in children, but its effectiveness is limited by the nature of the growth defect and the age of diagnosis. However, this treatment can have considerable side effects on carbohydrate metabolism, with some studies indicating increased fasting blood glucose and diabetes in children undergoing GH therapy (3).
GH administration is also an effective means of treating GH-deficient adults that suffer from increased fat mass, decreased lean muscle and muscle strength, and reduced exercise capacity. Exogenous GH treatment improves the metabolic profile, enhances the quality of life, increases lean muscle mass with a reduction in body fat, reduces total cholesterol, and increases lipolysis (4), but it can also lead to the deterioration of glucose metabolism (5). Overall, the use of exogenous GH for the treatment of GH deficiency is extremely useful but can have side effects or no additional benefits beyond particular dosages. Consequently, a method of amplifying the growth-promoting and anabolic effects of GH would be a very effective way to deal with this problem. One strategy would be to target or inactivate molecules that act to negatively regulate GH signaling.
The SOCS proteins are a family of negative regulatory proteins that are expressed in response to activation of a wide range of cytokine and growth factor signal cascades, particularly those that utilize JAK/STAT signaling systems. There are 8 known members (SOCS1–SOCS7 and cytokine-inducible SH2 domain–containing protein), and a number of these molecules have been shown, in in vitro overexpression systems, to bind to and inhibit a wide array of cytokine/growth factor signaling molecules, including GH (reviewed in 6, 7). Despite in vitro evidence that SOCS proteins display a high degree of promiscuity, clear physiological roles for a number of the SOCS members have been delineated through mouse genetic studies. SOCS1 has been shown to be involved in negatively regulating IFN responses (8), mammary gland function/prolactin signaling (9) and c-using cytokines (10), whereas SOCS3 plays central roles in placental development (11), as well as leukemia inhibitory factor (12), IL-6 (13-15), and granulocyte-CSF signaling (16).
Genetic studies have also shown that SOCS proteins play important roles in growth and development. SOCS2 knockout mice exhibit gigantism characterized by an increase in body weight and length, alterations in major urinary protein levels, thickening of dermal layers, and elevation of IGF-1 mRNA in some tissues (17). These phenotypes are observed in other models of excessive growth such as GH transgenic (18), IGF-1 transgenic (19) and high-growth mice (20), suggesting that SOCS2 may regulate some components of the somatotrophic pathway. While a number of studies have indicated that SOCS2 plays a role in GH signaling in vitro (21, 22), SOCS2 has also been found to bind to the IGF-1 receptor in yeast 2-hybrid studies (23, 24). We have also shown that STAT5b, one of the key mediators of GH action (25, 26), plays an important role in the development of the SOCS2–/– phenotype, and that modest prolongation of STAT5 signaling is evident in primary hepatocyte cultures of SOCS2-deficient mice stimulated with GH (27). Furthermore, using transgenic mice that overexpress a Flag-tagged SOCS2 construct, we have shown that SOCS2 interacts with endogenous GH receptors from a range of mouse tissues (28). Although the findings to date indicate that SOCS2 plays some role in the regulation of GH signaling, whether SOCS2 directly regulates GH signaling or IGF signaling, or both, remains an open question. A clear in vivo demonstration that the SOCS2-deficient phenotype relies upon the presence of GH would be invaluable in deciphering whether SOCS2 is a major negative regulator of GH action and would justify SOCS2 as a target for new therapeutic strategies.
Here we demonstrate that the presence of SOCS2 is dependent upon an intact somatotrophic pathway and that many aspects of the SOCS2–/– phenotype can be recapitulated with GH administration to mice lacking SOCS2 and endogenous GH. We further characterized the interaction between SOCS2 and the GH receptor and found that SOCS2 binds to 2 phosphorylated tyrosines on the GH receptor that are also targeted by another signaling regulator, Src homology 2–containing tyrosine phosphatase (SHP2), and these tyrosine residues are essential for the regulatory effects of SOCS2. Together, our data indicate that SOCS2 is a key modulator of GH sensitivity in vivo.
Results
Removal of GH deletes the SOCS2–/– phenotype.
Previous work with SOCS2-deficient mice has suggested that SOCS2 acts to negatively regulate the actions of GH (17, 27, 28). To test this hypothesis, we crossed male and female SOCS2–/– mice with Ghrhrlit/lit (little) mice, a mutant strain with a point mutation in the GH-releasing hormone receptor (GHRHR) that causes a nearly complete deficiency in pituitary-derived circulating GH and induces dwarfism (29). We used little mice because other models of GH deficiency or inaction do not exist (GH knockout mice), do not allow for GH growth recapitulation experiments (GH receptor knockout mice), or have other complicating deficiencies that could confound results (Ames and Snell dwarf mice). Growth curves were measured for Ghrhrlit/lit and SOCS2–/–Ghrhrlit/lit mice over a 12-week period (Figure 1). Female SOCS2–/–Ghrhrlit/lit mouse growth curves were indistinguishable from those of female Ghrhrlit/lit mice, whereas male SOCS2–/–Ghrhrlit/lit mice were only slightly larger than Ghrhrlit/lit mice by 12 weeks of age, suggesting that GH signaling is required for development of the SOCS2–/– phenotype.
Figure 1
GH is essential for the SOCS2–/– phenotype. Growth curves for SOCS2+/+Ghrhrlit/lit, SOCS2–/–Ghrhrlit/lit, SOCS2+/+GhrhrWT/WT and SOCS2–/–GhrhrWT/WT mice of both sexes over a 12-week period. At least 10–15 male mice were used at each point for the male growth curves and 6–17 female mice are represented at each time point for the female curves.
SOCS2 modulates growth responses to GH.
To definitively classify SOCS2 as a negative regulator of GH action, it is important not only to demonstrate dependence of the excess growth in SOCS2–/– mice on the presence of GH, but also to recapitulate the phenotype by supplying the cytokine exogenously to GH-deficient SOCS2–/– mice. Four-week-old male and female SOCS2+/+Ghrhrlit/lit and SOCS2–/–Ghrhrlit/lit mice were injected subcutaneously twice daily with 10 μg GH or saline for 4 weeks, and body weights were measured daily (Figure 2A). Growth was considerably exaggerated in male SOCS2–/–Ghrhrlit/lit mice receiving GH over that in male SOCS2+/+Ghrhrlit/lit mice receiving GH when evaluated against each genotype given saline injections (by 58% and 35%, respectively). This trend was also observed in SOCS2–/–Ghrhrlit/lit and SOCS2+/+Ghrhrlit/lit females given GH injections when evaluated against saline-treated mice (by 68% and 35%, respectively).
Figure 2
SOCS2 controls growth responses to GH. (A) Male and female SOCS2+/+Ghrhrlit/lit and SOCS2–/–Ghrhrlit/lit mice were weighed daily before being injected twice daily with 10 μg rpGH or saline for 28 days from 4 weeks of age. Growth curves were constructed from 5–10 male mice or 5–7 female mice per treatment group. (B) Differences in organ weights of male and female SOCS2+/+Ghrhrlit/lit (white bars) and SOCS2–/–Ghrhrlit/lit (black bars) mice are represented as a percentage increase over the mean of saline-injected mouse organ weights. *P < 0.05 vs. saline-injected mice; #P < 0.05 vs. SOCS2+/+Ghrhrlit/lit mice. Sal gland, salivary gland. (C) Picture of 6-month-old SOCS2–/–Ghrhrlit/lit mice that have had 3 pregnancies (upper) or no pregnancies (lower).
Analysis of organ and tissue weights from these mice further emphasized the role that SOCS2 plays in attenuating GH-driven growth (Figure 2B). The magnitude of GH-stimulated organ weight gain in male SOCS2–/–Ghrhrlit/lit mice was significantly greater than that observed in SOCS2+/+Ghrhrlit/lit mice in most tissues, particularly the carcass, which doubled in weight. The only tissue that decreased in weight was abdominal fat. Similar trends for organ weights were also observed in female SOCS2–/–Ghrhrlit/lit mice.
Another feature of the SOCS2–/– gigantic phenotype was the deposition of large amounts of collagen in ducts and vessels throughout the body, particularly in the skin (17). The enhanced growth response of SOCS2–/–Ghrhrlit/lit mice to GH was also reflected in modest increases in skin thickness of most male SOCS2–/–Ghrhrlit/lit mice at the end of GH-induced growth experiments (data not shown).
An interesting aspect of the female Ghrhrlit/lit mouse phenotype is the growth recovery observed throughout pregnancy that returns Ghrhrlit/lit females to wild-type size after 3 pregnancies (30). Although unmated SOCS2+/+Ghrhrlit/lit and SOCS2–/–Ghrhrlit/lit female mice were the same size, remarkably, following several pregnancies, SOCS2–/–Ghrhrlit/lit female mice not only reached wild-type female weight but attained the dimensions of adult SOCS2–/– female mice (Figure 2C and data not shown), indicating a hyperresponsiveness to a pregnancy-induced growth factor, such as prolactin or placental lactogens.
Increases in body weight were also accompanied by enlarged skeletal dimensions. Male SOCS2–/–Ghrhrlit/lit mice displayed enhanced body length compared to SOCS2+/+Ghrhrlit/lit mice upon GH treatment, and measurements of long bones revealed significant growth enhancement in SOCS2–/–Ghrhrlit/lit compared to SOCS2+/+Ghrhrlit/lit mice (Figure 3A). Computer tomography scanning of femurs from these mice revealed significant enhancement of a number of bone growth and strength parameters in male mice lacking SOCS2 (Figure 3B), but many of these changes were not observed in female mice (data not shown).
Figure 3
SOCS2 controls bone growth parameters. (A) Long bone lengths and femur morphometric data (B) were measured from 4–9 male mice of both genotypes. Data for each parameter is expressed as the percentage change with GH treatment compared to saline controls for SOCS2+/+Ghrhrlit/lit (white bars) and SOCS2–/–Ghrhrlit/lit (black bars) mice. *P < 0.05, vs. saline-treated mice; #P < 0.05 vs. SOCS2+/+Ghrhrlit/lit mice. Trabec. BMD, trabecular bone mineral density; circum., circumference.
GH-induced gene expression in livers of mice lacking SOCS2. While the studies described above provide good physiological evidence that SOCS2 attenuates GH signaling, we wished to examine whether a hyperresponsiveness to GH could be detected in the liver gene expression profiles of SOCS2–/–Ghrhrlit/lit mice compared to SOCS2+/+Ghrhrlit/lit mice, identify GH-responsive genes that are regulated by SOCS2, and gain insight into the molecular mechanism of SOCS2 action from the expression profile differences. We performed microarray analysis on liver RNA extracts from male SOCS2–/–Ghrhrlit/lit and SOCS2+/+Ghrhrlit/lit mice injected with GH 2 hours prior to sacrifice. We had previously shown that SOCS2 mRNA levels reach maximal concentrations 2 hours after GH treatment in the BRL4 hepatocyte cell line (31); therefore we supposed that differences in liver mRNA between SOCS2–/–Ghrhrlit/lit and SOCS2+/+Ghrhrlit/lit mice would be evident by this time. The number of genes induced and repressed by GH injection was increased almost 2-fold in the SOCS2–/–Ghrhrlit/lit livers compared to SOCS2+/+Ghrhrlit/lit livers (Figure 4A). Comparative analysis of genes with altered expression levels from both genotypes revealed considerable differences in the degree of change, with SOCS2–/–Ghrhrlit/lit mice demonstrating a hyperresponsiveness in nearly all cases (Figure 4B). Examination of the average change in gene expression of the SOCS2+/+Ghrhrlit/lit and SOCS2–/–Ghrhrlit/lit regulated gene populations also emphasizes the significant over-response of mice that lack SOCS2 (Figure 4C and Supplemental Table 1; supplemental material available online with this article; doi:10.1172/JCI200522710DS1). Hyperresponsiveness to GH is evident for well-known GH-regulated genes such as IGF-1, Spi2.2, fibrinogen, GADD45, and PPAR (32). Quantitative real-time RT-PCR analysis of the relative concentrations of 4 genes (IGF-1, PPAR, CYP8b1, and PIM-3) in the 2 models studied confirmed the results obtained by microarray analysis (Figure 4, D and E). Taken together, these results clearly demonstrate that the absence of SOCS2 creates a state of hypersensitivity to GH actions in the liver.
Figure 4
SOCS2 regulates GH-induced gene expression in the liver. (A) The number of GH-regulated genes in the liver of SOCS2+/+Ghrhrlit/lit and SOCS2–/–Ghrhrlit/lit mice 2 hours after GH injection was compared based on SAM analysis (5% FDR) for 4 independently replicated treatments. Individual genes are arranged along the x axis according to the value order of decreases and increases in gene expression of SOCS2–/–Ghrhrlit/lit mice. The y axis shows the log ratio of the transcript signals in GH-treated SOCS2+/+Ghrhrlit/lit and SOCS2–/–Ghrhrlit/lit mice (B), and the average changes in gene expression in SOCS2+/+Ghrhrlit/lit and SOCS2–/–Ghrhrlit/lit was also examined (C). Real-time RT-PCR results from SOCS2+/+Ghrhrlit/lit and SOCS2–/–Ghrhrlit/lit mouse livers showing genes that were downregulated (D) or upregulated (E). *P < 0.05.
Table 1
-screen analysis of SOCS2 SH2 domain protein interactions with receptor-derived phosphopeptides
All 3 domains of SOCS2 play crucial functions.
Given the evidence suggesting that SOCS2 negatively regulates GH signaling, we sought to understand the mechanism and nature of the SOCS2–GH receptor interaction. SOCS proteins have been defined as consisting of a central SH2 domain, an N-terminal domain of varying length, and a C-terminal motif termed the SOCS box (33, 34). While each of the 3 domains for SOCS1 and SOCS3 has been ascribed a function, little is known regarding the role each domain plays in SOCS2 regulation of GH function. Studies using SOCS2 overexpression systems have found that a low level of SOCS2 partially inhibits GH signaling, while higher concentrations cause a recovery and enhancement of signaling, implicating it as a dual effector molecule (35, 36). This hypothesis was strengthened with the observation that mice transgenically overexpressing SOCS2 at high levels suffered a mild excessive growth phenotype (28). Transient transfection assays, in which mutated SOCS2 proteins were expressed, were used to elucidate these roles (Figure 5A). SOCS2 that had a point mutation in a conserved arginine residue within the SH2 domain showed only slightly impaired inhibitory or enhancement effects, whereas the mutation of 2 additional sites next to this residue led to a more complete reduction of SOCS2 activity (Figure 5B). SOCS2 lacking the N terminus failed to have any significant effects on GH-mediated STAT5 reporter activity, but SOCS2 with the N terminus of SOCS1 substituted onto the molecule displayed some inhibitory capacity at higher concentrations of transfected construct (Figure 5C). A SOCS2 construct lacking the SOCS box displayed no inhibitory effects at all, but surprisingly triggered an enhancement of signaling even at low concentrations (Figure 5D). Other researchers have found that the SOCS-box motif from a range of SOCS molecules binds Elongin B and C complexes (37, 38), and we found an association between SOCS2 and Elongin B and C in immunoprecipitation studies (Figure 5E).
Figure 5
SOCS2 motif control of GH signaling. (A) A schematic diagram of SOCS2 is provided to help clarify mutant constructs used and residues mutated in the SH2 domain. (B–D) 293T cells were transfected with pig GH receptor and SOCS2 (SOCS2 WT), or with the following SOCS2 mutant constructs: (B) SOCS2 with a point mutation in the SH2 domain R73K (SOCS2 D) or SOCS2 with a triple mutation at R73K, D74E, and S75C in the SH2 domain (SOCS2 KD); (C) SOCS2 lacking the 37-AA N terminus (SOCS2°NT) or SOCS2 with the N-terminal region of SOCS1 (SOCS1/2/2); or (D) SOCS2 lacking the 39-AA C terminus (SOCS2°SB) at a range of plasmid concentrations (ng). The transfected cells were then stimulated with rpGH and the luciferase activity from an LHRE-luciferase reporter was measured. Data is corrected for transfection efficiency by cotransfection of a ?-galactosidase–expressing plasmid. Luciferase activity was corrected using values obtained in the absence of GH, then expressed as a percentage of wild-type activity, which was assigned a value of 100%. Experiments were performed in triplicate, and data presented here are representative of 3 independent experiments. (E) Flag-tagged SOCS2 and empty vector were transfected into 293T cells, lysed, and immunoprecipitated using antibodies against Flag. After separation on SDS-PAGE and Western transfer, blots were probed with antibodies against Elongins B and C, then stripped and reprobed with antibodies against the Flag epitope.
SOCS2 actions are dependent upon specific sites on the GH receptor.
We have previously shown that SOCS2 interacts with endogenous GH receptor from a number of different mouse tissues and that this interaction occurs at Tyr595 in a phosphorylation-dependent manner (28). Modification of the recombinant SOCS2 protein purification strategy to use cobalt metal ion affinity resin and thrombin to enzymatically cleave NusA from SOCS2 protein led to a significant stabilization of protein activity (data not shown), thus allowing a more thorough examination of SOCS2–GH receptor interactions.
Biomolecular interaction analysis, using recombinant SOCS2 SH2 domain protein passed over phosphorylated peptides derived from the GH receptor fixed to a biosensor chip, confirmed the Tyr595 interaction and its dependence on phosphorylation, but also indicated that an additional residue, Tyr487, is also a site of interaction (Figure 6). Interestingly, Tyr595 and Tyr487 have previously been implicated as SHP2 binding sites that regulate GH receptor signaling (39). Biomolecular interaction analysis of recombinant SHP2 protein binding to GH receptor–derived peptides confirmed Tyr595 and Tyr487 interactions, but also indicated some binding with the Tyr534 residue (Figure 6). Verification of SOCS2-GH receptor peptide interactions was performed using an AlphaScreen (PerkinElmer Life Sciences) interaction assay (40, 41). The -screen assay utilizes donor beads coated with phosphorylated peptide and acceptor beads coated with recombinant SOCS2-SH2 domain protein. Interaction between phosphopeptide and SOCS2 brings the acceptor and donor beads into close proximity, and excitation of the donor beads by laser light (680 nm) induces production of highly reactive singlet oxygen that diffuses from the donor bead for a distance of approximately 200 nm in aqueous solution before rapidly decaying. When an acceptor bead is held in close proximity to the donor bead, singlet oxygen reacts with a reagent in the acceptor beads to generate chemiluminescence of 580 to 620 nm. The intensity of the fluorescence output therefore provides a quantifiable measurement of the SOCS2/phosphopeptide binding interaction. Analysis of SOCS2/peptide interactions using the -screen assay showed that phosphorylated Tyr595 and Tyr487 peptides bound to the SOCS2 SH2 domain with high affinity compared to Tyr332, Tyr534, and nonphosphorylated Tyr595 (Table 1).
Figure 6
SOCS2 interacts with tyrosine residues on the GH receptor. Biosensor analysis was performed on the binding between GH receptor–derived phosphopeptides and NusA.SOCS2 SH2 protein (A) or SHP2 protein (B). Sensorgrams correspond to binding of immobilized peptide on a streptavidin sensorchip.
To validate the biological relevance of the candidate GH receptor interaction sites, we generated GH receptor constructs that lacked one or more of the tyrosine residues identified by biomolecular interaction analysis and used these in transfection studies. The activity of these constructs was examined by their ability to stimulate the STAT5-responsive luciferase reporter with and without GH stimulation (Figure 7A). Mutant receptors that lacked either Tyr487 or Tyr595 demonstrated enhanced reporter activity, and receptors lacking both showed a compounded increase in activity.
Figure 7
SOCS2 effects are mediated through Tyr487 and Tyr595. (A) 293T cells were transfected with wild-type or mutated GH receptor constructs, then they were starved of (white bars) or stimulated with rpGH (black bars), and the luciferase activity from an LHRE-luciferase reporter was measured. Data were corrected for transfection efficiency by cotransfection of a ?-galactosidase–expressing plasmid. *P < 0.05, significant difference between stimulated receptors. (B–D) 293T cells were transfected with wild-type GH receptor, (B) Y595F GH receptor, (C) Y487F GH receptor, or (D) Y487,595F GH receptor and increasing concentrations of SOCS2 plasmid (ng). Data were corrected for transfection efficiency by cotransfection of a ?-galactosidase–expressing plasmid. Luciferase activity was corrected using values obtained in the absence of GH, then expressed as a percentage of wild-type GH receptor activity without SOCS2 expression, which was assigned a value of 100%. Experiments were performed in triplicate and data presented here are representative of 3 independent experiments. Expression of SOCS2 was confirmed by Western blotting of cell lysate with antibodies against the Flag epitope at the N terminus of the SOCS2 expression construct.
However, expression of the data in terms of fold induction after GH stimulation failed to reflect these changes (14.7-fold for wild-type receptor, 13.8-fold for Y487F receptor, 16.2-fold for Y595F receptor, and 14.6-fold for Y487,595F receptor). Real-time PCR was also used to determine that only very low levels of endogenous SOCS2 were present in 293T cells when compared to exogenous SOCS2 expression levels, and this low level of endogenous SOCS2 probably did not contribute to the upregulation of STAT5 activation by the Y487,595F mutant GH receptor in the unstimulated state (data not shown).
Titrations of SOCS2 have previously demonstrated a biphasic response in terms of GH-stimulated STAT5 reporter activity derived from wild-type GH receptors: low levels of SOCS2 inhibited approximately 50% of signaling, whereas higher levels led to a recovery or enhancement of signal (28). Consequently, it was expected that removal of SOCS2 binding sites would ameliorate these effects. Analysis of the Y487F mutant construct revealed that SOCS2 could still inhibit signaling by this receptor, although the degree of recovery may have lessened (Figure 7C). However, there appeared to be some weakening of the inhibitory effects of SOCS2 on the Y595F mutant (Figure 7B). Deletion of both the Tyr487 and Tyr595 residues removed all the inhibitory effects of SOCS2 and also significantly affected the recovery effects of SOCS2 (Figure 7D). Analysis of Tyr534 mutant SOCS2 revealed no significant role for this residue in the ability of SOCS2 to function (data not shown).
Discussion
The amelioration and reconstitution of the excessive growth phenotype in SOCS2–/– mice in response to GH provides compelling evidence that SOCS2 plays an important role in the negative regulation of GH signaling. The exact site of SOCS2 action in the somatotrophic axis has been controversial, as SOCS2 has been shown to bind to the IGF-1 receptor in yeast 2-hybrid studies (23), and the phenotype of SOCS2 knockout mice has some similarities to that of IGF-1 transgenic mice (19). However, there are no data to indicate that IGF-1 induces SOCS2 mRNA expression in vitro, and IGF-1 signaling is not perturbed in SOCS2-deficient embryonic fibroblasts (27). The hypothesis that SOCS2 is a negative regulator of GH signaling is supported by data presented here and published results showing the following: SOCS2-GH receptor interactions in vitro and in vivo (28); the role of STAT5b in the development of the SOCS2–/– phenotype; modestly prolonged GH signaling in SOCS2-deficient hepatocytes (27); and increases in size of non–IGF-1 responsive tissues such as the liver (17, 27). Furthermore, detection of significant differences in the liver gene expression profiles in the SOCS2+/+Ghrhrlit/lit and SOCS2–/–Ghrhrlit/lit mice 2 hours after GH injection almost certainly precludes the role of other growth factors in mediating these differences. We do not believe that there is sufficient time for GH signaling to induce another growth factor to then act upon its own signaling system to induce SOCS2 mRNA expression, and then for the subsequent protein to exert an effect such that it could be detected in mRNA.
The hyperresponsiveness of these genes to GH in hepatic tissue, as well as the identification of new GH-regulated genes in the double knockout, further emphasize that SOCS2 is an important regulator of GH signaling and a therapeutic target for the enhancement of the growth-promoting effects of GH. The magnitude of organ and tissue growth in SOCS2-deficient mice in response to GH was significant, but size differences were also tissue dependent. Tissue-specific sensitivities to GH, the level and timing of SOCS2 expression in that tissue, gender, and sexual dimorphisms all appeared to be modifying factors in controlling target sensitivity to GH. This is emphasized in the pregnancy-driven enhancement of body size in SOCS2–/–Ghrhrlit/lit females. Although the mechanism behind this phenomenon is not completely clear, it is plausible that pregnant female mice are exposed to placentally derived GH or prolactin, to which SOCS2–/–Ghrhrlit/lit mice are hyperresponsive.
The unique nature of SOCS2 action is further emphasized in the microarray studies. SOCS2–/–Ghrhrlit/lit mice clearly have an enhanced GH-induced liver gene expression response compared to SOCS2+/+Ghrhrlit/lit mice, although the magnitude and type of response do not simply fit a model of a completely deregulated GH receptor signaling action. IGF-1 is a classic GH-induced gene of the liver, and it has been shown to be a contributor to somatic growth (42). The overexpression of IGF-1 in SOCS2–/–Ghrhrlit/lit mice may contribute to their increased growth response when administered GH. The appearance of other GH-regulated genes such as PPAR and Spi2.2 fits the proposed model, but the alterations of other genes that were previously thought to be unresponsive to GH (e.g., PIM-3 and CYP8b1) indicate that SOCS2 function is more complicated than first thought. Similar observations have been made in other SOCS knockout models where IL-6 stimulation of SOCS3-deficient livers led to exaggerated gene expression of IL-6–responsive genes, as compared to wild-type expression, but it also let to the induction of other genes thought to be classically IFN-–inducible genes (12, 14). The magnitude of change in GH-induced expression with a lack of SOCS2 was not as large as might be predicted from that observed in SOCS3-deficient livers stimulated with IL-6 (14), but these small changes in signaling and expression may be large when considering that they would be cumulative over an animal’s somatic growth period.
Our results demonstrate that all 3 domains of SOCS2 may play a role in protein function. The SH2 domain of a number of SOCS proteins plays a central role in their function and SOCS2 is no different. Whereas mutating the conserved arginine in the SOCS1 SH2 domain was sufficient to eliminate the inhibitory activity of SOCS1 (43), SOCS2 required additional mutations to become inactive. The reasons for this are unclear, but it may reflect redundancy in SOCS2 generated by its ability to bind a number of different residues on the GH receptor. The SOCS box of SOCS2 plays a central role in the negative regulation of GH signaling. This is intriguing, as removal of the SOCS box from other SOCS molecules does not impede inhibitory function in overexpression studies, although the SOCS box of SOCS1 has been shown to play an important role physiologically (44). Consequently, this may reflect a difference in the mechanism of SOCS2 action, with a strong reliance on ubiquitination and degradation to terminate GH signaling, rather than on inhibition of receptor function. Consistent with this interpretation, SOCS1 and SOCS3 contain a kinase inhibitory region that is absent in SOCS2 (45).
We have shown here that it is necessary to delete both the Y487 and Y595 residues to remove the inhibitory effects of SOCS2 on GH signaling. Mutations of these 2 sites have previously been shown to result in the prolongation of the GH-activated JAK/STAT5 pathway. SHP2 binds these residues and is thought to act as the negative regulator of the receptor (39). However, when the function of SHP2 is directly tested in overexpression systems, it acts as a positive regulator of GH signaling (46). In retrospect, the data supporting a role for SHP2 as a negative regulator generated using GH receptor tyrosine mutations could now be reinterpreted as SOCS2 mediating the inhibition directly or acting to inhibit SHP2 action (39). This model has a number of similarities to that for SOCS3 and SHP2 binding to the gp130 receptor (47-49). The observations of significant increases in luciferase output of the GH mutant receptors when stimulated with hormone, as well as increased basal levels without stimulation, are also seen in similar studies in which the SOCS3 binding site of gp130 was mutated (48). A possible explanation for this phenomenon is that basal receptor activity also utilizes low levels of SOCS2 for negative control, and mutation of SOCS2 binding residues causes a proportional increase in luciferase output compared to the stimulated levels. Presuming that SOCS2 protein levels are low in unstimulated cells, it is possible that other negative regulators can interact with the receptor through these tyrosines. Another candidate is PTP1B, a tyrosine phosphatase known to interact with the GH receptor and has been confirmed to have negative actions on GH signaling (50). A negative action of SHP2 on certain tissues cannot be excluded, and the fact that SOCS2 and SHP2 both bind the same sites on the GH receptor sets the scene for potential interplay between these molecules. It is anticipated that further characterization of these phosphatases and SOCS2 interactions in the negative regulation of the GH receptor will decipher the nature of interplay that may exist between these molecules and provide further insights into the exact mechanism by which SOCS2 inhibits GH signaling.
Converting the information gathered in this manuscript into a clinical application is the next step. The -screen assay technology used in this work forms the basis of a high-throughput screen, and we are currently using this approach to screen chemical compound libraries for small-molecule inhibitors of SOCS2-GH receptor interactions. An alternate approach would be to downregulate SOCS2 mRNA expression by using antisense or siRNA approaches, which are becoming more prevalent in strategies to target intracellular molecules. It is hoped that such approaches may be useful in the development of new strategies targeting growth disorders by replacing or amplifying the effects of exogenous GH administration.
Methods
Animals.
Ghrhrlit/lit mice were obtained from Jackson ImmunoResearch Laboratories and were maintained by mating Ghrhrlit/lit or GhrhrWT/lit females with Ghrhrlit/lit males. Mice were phenotyped for the little mutation after weaning. SOCS2–/– mice were mated with Ghrhrlit/lit mice and genotyped for SOCS2 as previously described (17), while genotyping of the little mutation was performed by PCR across the mutation and direct sequencing of PCR products (51). SOCS2–/–Ghrhrlit/lit mice were generated from matings between male SOCS2–/–Ghrhrlit/lit or SOCS2–/–Ghrhrlit/WT mice and female SOCS2–/–Ghrhrlit/lit or SOCS2–/–Ghrhrlit/WT mice. All experiments were performed in accordance with National Health and Medical Research Council of Australia guidelines and were approved by the Melbourne Health Research Directorate Animal Ethics Committee (Melbourne, Victoria, Australia).
For GH-induced growth experiments, 10 μg of recombinant pig GH (rpGH) dissolved in 50 μl saline, or 50 μl saline alone, was injected subcutaneously twice daily on weekdays and once daily on weekends. The site of injection was rotated daily to minimize discomfort, and weight measurements were taken in the morning before injection.
Microarray experiments were performed on livers from male SOCS2+/+Ghrhrlit/lit and SOCS2–/–Ghrhrlit/lit mice that had been injected with 100 μg rpGH 2 hours before sacrifice.
Histological examination.
Skin sections were prepared from tissues fixed in 10% saline-buffered formalin by standard techniques, stained with hematoxylin and eosin, and examined by light microscopy. Bone lengths were measured using x-ray imaging as previously described (17). Other bone measurements were obtained by computerized tomography using a Stratec pQCT XCT Research M (Norland; v5.4B) operating at a resolution of 70 μm as previously described (52). Trabecular bone mineral density was determined ex vivo, with a metaphyseal pQCT scan of the distal femur and the proximal tibia. The scan was positioned in the metaphysis at a distance corresponding to 4% of the total length of either the femur or the tibia. The trabecular bone region was defined as the inner 45% of the total cross-sectional area. Cortical bone parameters were determined ex vivo with a mid-diaphyseal pQCT scan of the femur and tibia.
Elongin B and C interaction.
Either pEF-Flag or pEF-SOCS2-Flag plasmids (200 ng) (43) was transfected into 293T human embryonic kidney cells. Cells were washed and lysed 48 hours after transfection, and lysates were precipitated with antibodies against the Flag epitope (monoclonals 9H1 and 9B4 provided by David Huang, WEHI). After SDS-PAGE separation and Western transfer, blots were probed with antibodies against Elongin B and C (provided by Zhang-Guo Jiang, WEHI) then reprobed with antibodies directed against the Flag epitope.
Vector construction.
Mutations in the SH2 domain of SOCS2 were generated using splice overlap extension PCR of the SOCS2R73K plasmid (43) to generate additional mutations, D74E and S75C. SOCS2°box (lacking the last 39 amino acids) and SOCS2°Nterm (lacking the first 37 amino acids) constructs were generated by PCR to give fragments with in-frame AscI and MluI restriction enzyme sites at the N and C termini, respectively, and subcloned into pEF-Flag-I to give proteins with an N-terminal Flag epitope tag. A plasmid encoding the N terminus of SOCS1 (amino acids 1–78) fused to the SOCS2°Nterm was generated using PCR-based techniques as previously described (43). GH receptor constructs for mutational analysis were generated by excising the GH receptor coding region from the pMet-Ig7 GHR plasmid (sourced from Nils Billestrup, Steno Diabetes Center, Gentofte, Denmark ) and inserting it into the pEF-BOS vector. The Y487F, Y595F, Y534F, and Y487,595F mutants were generated by using overlapping PCR strategies to introduce mutations at these sites. All constructs were sequenced in their entirety before use.
Luciferase assays.
293T cells were transfected with Lipofectamine 2000 (Invitrogen Corp.) reagents according to the manufacturer’s instructions. For SOCS2 domain analysis, 4 x 105 cells were transfected with 200 ng each of pIG-GH receptor plasmid, lactogenic hormone response element–luc (LHRE-luc) reporter plasmid, ?-galactosidase plasmid, and 0–200 ng pEF-SOCS2-Flag plasmid (43) made up to 200 ng with pEF-BOS plasmid into a 24-well plate 6 hours after cells were plated. Twenty-four hours later, cells were washed once with mouse-tonicity PBS (18.9 mM Na2HPO4, 3.9 mM NaH2PO4 x H2O, and 148 mM NaCl) and the culture medium was replaced with DMEM containing 1% BSA. Cells were left to equilibrate for 1 hour before 500 ng/ml of rpGH was added to appropriate groups. Cells were incubated for a further 16 hours before being lysed and assayed for luciferase and ?-galactosidase activity as described (28). Luciferase activity was normalized to ?-galactosidase activity. Luciferase assays examining point mutations in GH receptors were performed as described above except that only 2 ng of each pEF-GH receptor plasmid was used.
Recombinant protein expression.
Murine SOCS2 SH2 domain was produced as described previously (28), with the following modifications. Bacterial lysate containing NusA-hexaHis-SOCS2-SH2 fusion protein was passed through a Talon column (cobalt metal ion affinity chromatography ) to bind fusion protein before the NusA protein was cleaved from the column at a thrombin cleavage site using thrombin (Sigma-Aldrich). The column was washed extensively with PBS (pH 6.4) before the SOCS2 SH2 domain was eluted with 150 mM imidazole in PBS (pH 7.5). Fractions were collected and separated by SDS-PAGE to determine purity. Recombinant SHP2 protein was produced as described previously (48).
SDS-PAGE analysis and Western blotting.
All SDS-PAGE, Western blotting, and detection of Flag-tagged proteins was performed as described previously (27, 28).
Biomolecular interaction analysis.
All analyses were performed essentially as previously described for SOCS3 (48). Briefly, biotinylated phosphopeptides (1 μg/ml) were bound to streptavidin-coated biosensor chips (SA5, Biacore) at a density of 400 RU. SOCS2 binding studies were measured in buffer A (20 mM HEPES, 150 mM sodium chloride, 3.4 mM EDTA, and 0.005% (v/v) Tween 20) with a flow rate of 10 μl/min. Chips were washed with 6 M guanidinium hydrochloride between binding measurements to remove residual SOCS2 protein, then washed with buffer A. Binding profiles were analyzed using BIAEVALUATION software Ver. 3.0 (Pharmacia).
AlphaScreen binding assay.
Nickel chelate–derived AlphaScreen beads (PerkinElmer Life Sciences) were diluted to a final concentration of 5 mg/ml in buffer (bead buffer) containing 0.1 mg/ml casein, 50 mM HEPES (pH 7.4), 10 mM dithithreitol, 100 mM NaCl, and 0.1% Tween 20. Hexa-His–tagged SOCS2-SH2 domain was added at a final concentration of 16 nM and the mixture was incubated for 30 minutes at room temperature. The biotinylated phosphopeptide was mixed at a concentration of 32 nM with 5 mg/ml of streptavidin-coated -screen beads in bead buffer and incubated at room temperature for 30 minutes. The assay was performed by mixing 10 ml of each bead stock solution in the wells of a 384-well microtiter plate and incubating the mixture at room temperature for 2.5 hours. The assay results were read using a Fusion Alpha plate reader (PerkinElmer Life Sciences). For peptide competition experiments, the non-biotinylated competitor peptide was diluted in bead buffer and 5 ml of this stock solution was mixed with 10 ml of the SOCS2 bead solution in the wells of the microtiter plate. The mixture was incubated for 30 minutes at room temperature before 10 ml of streptavidin beads coated with 32 nM of Tyr595 was added. The rest of the assay was performed as described above.
Microarray analysis.
A microarray containing 16,500 mouse oligo 70-mers, printed twice and produced at the SweGene DNA Microarray Resource Center at Lund University, was used to evaluate the hepatic GH responsiveness in the 2 models studied. Total RNA was isolated by homogenization of liver tissue using TRIzol Reagent (Invitrogen Corp.), according to the protocol supplied by the manufacturer. Twelve independent hybridizations were performed, comparing individual animals from the different experimental groups (SOCS2+/+Ghrhrlit/lit vs. SOCS2–/–Ghrhrlit/lit, SOCS2+/+Ghrhrlit/lit vs. SOCS2+/+Ghrhrlit/lit + GH, and SOCS2–/–Ghrhrlit/lit vs. SOCS2–/–Ghrhrlit/lit + GH). The protocols employed for probe labeling, purification, and hybridization were essentially as described previously (32). Total RNA (20 μg) was used in each of the labeling reactions and balance dye swaps were included in the experimental design. Image analysis was performed using GenePix Pro software (Axon Instruments). Fluorescence ratios were normalized (54) using the locally weighted scatter plot smoother (LOWESS) method in the statistics for microarray (SMA) package (Axon Instruments). Individual measurements for specific probes were calculated as the gene average of 2 probe measurements. The variability of the analysis was estimated using significance analysis of microarray (SAM) software (Stanford University) (55). A q value was assigned for each of the detectable genes in the array. This value is similar to the P values, measuring the lowest false discovery rate (FDR) at which the differential expression (the ratio between control and treatment) of the gene is called significant. In this study, genes with a FDR value of less than 5% were listed as differentially expressed. In order to allow for comparison between models, the analysis was performed on gene probes that were consistently detected in both models (i.e., in 3 out of the 4 independent measurements). A total of 2,219 gene-specific probes were used for the comparison. Statistical analysis comparing the average of log ratio values in both models was performed using Student’s t test analysis; complete results are available at http://www.cmm.ki.se/gnorstedt/CCMCore/Index.html.
Gene expression analysis by real-time quantitative RT-PCR.
The expression of IGF-1, PPAR-, CYP8b1 (12 hydroxylase), and the serine/threonine kinase PIM-3 was analyzed using quantitative real-time PCR (DNA Engine Opticon 2 System; MJ Research Inc.) in RNA samples from individual animals. Briefly, 2 μg of total RNA treated with DNase I (Promega) was reverse-transcribed using a First Strand cDNA Synthesis Kit (Invitrogen Corp.). Gene-specific primers corresponding to the target genes were used to generate the amplicons.
To measure the relative concentration of the gene expression, 2 μl of each sample (dilution 1:3) was analyzed in duplicate. Real-time PCR was performed in a 20 μl volume with 2 μl of respective cDNA and 0.4 μM primers. Nucleotides, Taq polymerase, reaction buffer, and SYBR Green I dye were supplied in the iQ SYGR Green Supermix (Bio-Rad Laboratories Inc.). The amplification program consisted of 1 cycle of 95°C for 3 minutes, followed by 45 cycles of 95°C for 15 seconds, 10 seconds at annealing temperature, and 72°C for 30 seconds; fluorescent intensity was measured at a specific acquisition temperature for each gene. The individual mRNA levels were obtained by comparison to a standard curve. GH-induced changes in expression were calculated after normalization with the level of ?-actin in each sample.
Acknowledgments
We thank Sally Cane and Janelle Lochlan for genotyping, Steven Mihajlovic for histology, Megan James for excellent animal husbandry, and Zdenka Bolcevic for comments on the manuscript. This work is supported by the Australian National Health and Medical Research Council (program grant 257500), the Australian Federal Government Cooperative Research Centres Program, the NIH (grant CA22556), Amrad Corporation Ltd., the Swedish Research Council (grant 529-2002-6766), the Wallenberg Foundation, and the Swedish Society for Medical Research. C.J. Greenhalgh is the recipient of an Australian Postdoctoral Research Fellowship.
References
Press, M. 1988. . Growth hormone and metabolism. Diabetes Metab. Rev. 4::391-414.
Sonksen, P.H., Salomon, F., and Cuneo, R. 1991. . Metabolic effects of hypopituitarism and acromegaly. Horm. Res. 36::S27-S31.
. 1998. . Consensus guidelines for the diagnosis and treatment of adults with growth hormone deficiency: summary statement of the growth hormone research society workshop on adult growth hormone deficiency. J. Clin. Endocrinol. Metab. 83::379-381.
Monson, J.P. 2003. . Long-term experience with GH replacement therapy: efficacy and safety. Eur. J. Endocrinol. 148::S9-S14.
Bramnert, M. et al. 2003. . Growth hormone replacement therapy induces resistance by activating the glucose-fatty acid cycle. J. Clin. Endocrinol. Metab. 88::1455-1463.
Hanada, T., Kinjyo, I., Inagaki-Ohara, K., and Yoshimura, A. 2003. . Negative regulation of cytokine signaling by CIS/SOCS family proteins and their roles in inflammatory diseases. Rev. Physiol. Biochem. Pharmacol. 149::72-86.
Alexander, W.S. 2002. . Suppressors of cytokine signalling (SOCS) in the immune system. Nat. Rev. Immunol. 2::410-416.
Alexander, W.S. et al. 1999. . SOCS1 is a critical inhibitor of interferon gamma signaling and prevents the potentially fatal neonatal actions of this cytokine. Cell. 98::597-608.
Lindeman, G.J. et al. 2001. . SOCS1 deficiency results in accelerated mammary gland development and rescues lactation in prolactin receptor-deficient mice. Genes Dev. 15::1631-1636.
Chong, M.M. et al. 2003. . Suppressor of cytokine signaling-1 is a critical regulator of interleukin-7-dependent CD8+ T cell differentiation. Immunity. 18::475-487.
Roberts, A.W. et al. 2001. . Placental defects and embryonic lethality in mice lacking suppressor of cytokine signaling 3. Proc. Natl. Acad. Sci. U. S. A. 98::9324-9329.
Lang, R. et al. 2003. . SOCS3 regulates the plasticity of gp130 signaling. Nat. Immunol. 4::546-550.
Takahashi, Y. et al. 2003. . SOCS3: an essential regulator of LIF receptor signaling in trophoblast giant cell differentiation. EMBO J. 22::372-384.
Croker, B.A. et al. 2003. . SOCS3 negatively regulates IL-6 signaling in vivo. Nat. Immunol. 4::540-545.
Yasukawa, H. et al. 2003. . IL-6 induces an anti-inflammatory response in the absence of SOCS3 in macrophages. Nat. Immunol. 4::551-556.
Croker, B.A. et al. 2004. . SOCS3 is a critical physiological negative regulator of G-CSF signaling and emergency granulopoiesis. Immunity. 20::153-165.
Metcalf, D. et al. 2000. . Gigantism in mice lacking suppressor of cytokine signalling-2. Nature. 405::1069-1073.
Palmiter, R.D. et al. 1982. . Dramatic growth of mice that develop from eggs microinjected with metallothionein-growth hormone fusion genes. Nature. 300::611-615.
Mathews, L.S. et al. 1988. . Growth enhancement of transgenic mice expressing human insulin-like growth factor I. Endocrinology. 123::2827-2833.
Horvat, S., and Medrano, J.F. 2001. . Lack of Socs2 expression causes the high-growth phenotype in mice. Genomics. 72::209-212.
Hansen, J.A., Lindberg, K., Hilton, D.J., Nielsen, J.H., and Billestrup, N. 1999. . Mechanism of inhibition of growth hormone receptor signaling by suppressor of cytokine signaling proteins. Mol. Endocrinol. 13::1832-1843.
Ram, P.A., and Waxman, D.J. 1999. . SOCS/CIS protein inhibition of growth hormone-stimulated STAT5 signaling by multiple mechanisms. J. Biol. Chem. 274::35553-35561.
Dey, B.R., Spence, S.L., Nissley, P., and Furlanetto, R.W. 1998. . Interaction of human suppressor of cytokine signaling (SOCS)-2 with the insulin-like growth factor-I receptor. J. Biol. Chem. 273::24095-24101.
Lopaczynski, W. 1999. . Differential regulation of signaling pathways for insulin and insulin-like growth factor I. Acta Biochim. Pol. 46::51-60.
Udy, G.B. et al. 1997. . Requirement of STAT5b for sexual dimorphism of body growth rates and liver gene expression. Proc. Natl. Acad. Sci. U. S. A. 94::7239-7244.
Teglund, S. et al. 1998. . Stat5a and Stat5b proteins have essential and nonessential, or redundant, roles in cytokine responses. Cell. 93::841-850.
Greenhalgh, C.J. et al. 2002. . Growth enhancement in suppressor of cytokine signaling 2 (SOCS-2)-deficient mice is dependent on signal transducer and activator of transcription 5b (STAT5b). Mol. Endocrinol. 16::1394-1406.
Greenhalgh, C.J. et al. 2002. . Biological evidence that SOCS-2 can act either as an enhancer or suppressor of growth hormone signaling. J. Biol. Chem. 277::40181-40184.
Wajnrajch, M.P., Gertner, J.M., Harbison, M.D., Chua, S.C. Jr., and Leibel, R.L. 1996. . Nonsense mutation in the human growth hormone-releasing hormone receptor causes growth failure analogous to the little (lit) mouse. Nat. Genet. 12::88-90.
Beamer, W.H., and Eicher, E.M. 1976. . Stimulation of growth in the little mouse. J. Endocrinol. 71::37-45.
Flores-Morales, A. et al. 2001. . Endoplasmic reticulum stress prolongs GH-induced Janus kinase (JAK2)/signal transducer and activator of transcription (STAT5) signaling pathway. Mol. Endocrinol. 15::1471-1483.
Tollet-Egnell, P. et al. 2001. . Gene expression profile of the aging process in rat liver: normalizing effects of growth hormone replacement. Mol. Endocrinol. 15::308-318.
Hilton, D.J. et al. 1998. . Twenty proteins containing a C-terminal SOCS box form five structural classes. Proc. Natl. Acad. Sci. U. S. A. 95::114-119.
Kile, B.T. et al. 2002. . The SOCS box: a tale of destruction and degradation. Trends Biochem. Sci. 27::235-241.
Favre, H., Benhamou, A., Finidori, J., Kelly, P.A., and Edery, M. 1999. . Dual effects of suppressor of cytokine signaling (SOCS-2) on growth hormone signal transduction. FEBS Lett. 453::63-66.
Adams, T.E. et al. 1998. . Growth hormone preferentially induces the rapid, transient expression of SOCS-3, a novel inhibitor of cytokine receptor signaling. J. Biol. Chem. 273::1285-1287.
Kamura, T. et al. 1998. . The Elongin BC complex interacts with the conserved SOCS-box motif present in members of the SOCS, ras, WD-40 repeat, and ankyrin repeat families. Genes Dev. 12::3872-3881.
Zhang, J.G. et al. 1999. . The conserved SOCS box motif in suppressors of cytokine signaling binds to elongins B and C and may couple bound proteins to proteasomal degradation. Proc. Natl. Acad. Sci. U. S. A. 96::2071-2076.
Stofega, M.R., Herrington, J., Billestrup, N., and Carter-Su, C. 2000. . Mutation of the SHP-2 binding site in growth hormone (GH) receptor prolongs GH-promoted tyrosyl phosphorylation of GH receptor, JAK2, and STAT5B. Mol. Endocrinol. 14::1338-1350.
Ullman, E.F. et al. 1994. . Luminescent oxygen channeling immunoassay: measurement of particle binding kinetics by chemiluminescence. Proc. Natl. Acad. Sci. U. S. A. 91::5426-5430.
Ullman, E.F. et al. 1996. . Luminescent oxygen channeling assay (LOCI): sensitive, broadly applicable homogeneous immunoassay method. Clin. Chem. 42::1518-1526.
Yakar, S. et al. 2002. . Circulating levels of IGF-1 directly regulate bone growth and density. J. Clin. Invest. 110::771-781. doi:10.1172/JCI200215463.
Nicholson, S.E. et al. 1999. . Mutational analyses of the SOCS proteins suggest a dual domain requirement but distinct mechanisms for inhibition of LIF and IL-6 signal transduction. EMBO J. 18::375-385.
Zhang, J.G. et al. 2001. . The SOCS box of suppressor of cytokine signaling-1 is important for inhibition of cytokine action in vivo. Proc. Natl. Acad. Sci. U. S. A. 98::13261-13265.
Yasukawa, H. et al. 1999. . The JAK-binding protein JAB inhibits Janus tyrosine kinase activity through binding in the activation loop. EMBO J. 18::1309-1320.
Kim, S.O., Jiang, J., Yi, W., Feng, G.S., and Frank, S.J. 1998. . Involvement of the Src homology 2-containing tyrosine phosphatase SHP-2 in growth hormone signaling. J. Biol. Chem. 273::2344-2354.
Schmitz, J., Weissenbach, M., Haan, S., Heinrich, P.C., and Schaper, F. 2000. . SOCS3 exerts its inhibitory function on interleukin-6 signal transduction through the SHP2 recruitment site of gp130. J. Biol. Chem. 275::12848-12856.
Nicholson, S.E. et al. 2000. . Suppressor of cytokine signaling-3 preferentially binds to the SHP-2-binding site on the shared cytokine receptor subunit gp130. Proc. Natl. Acad. Sci. U. S. A. 97::6493-6498.
Fairlie, W.D., De Souza, D., Nicola, N.A., and Baca, M. 2003. . Negative regulation of gp130 signalling mediated through tyrosine-757 is not dependent on the recruitment of SHP2. Biochem. J. 372::495-502.
Gu, F. et al. 2003. . Protein tyrosine phosphatase 1B attenuates growth hormone-mediated JAK2-STAT signaling. Mol. Cell Biol. 23::3753-3762.
Godfrey, P. et al. 1993. . GHRH receptor of little mice contains a missense mutation in the extracellular domain that disrupts receptor function. Nat. Genet. 4::227-232.
Windahl, S.H., Vidal, O., Andersson, G., Gustafsson, J.A., and Ohlsson, C. 1999. . Increased cortical bone mineral content but unchanged trabecular bone mineral density in female ER?–/– mice. J. Clin. Invest. 104::895-901.
Hansen, L.H. et al. 1996. . Identification of tyrosine residues in the intracellular domain of the growth hormone receptor required for transcriptional signaling and Stat5 activation. J. Biol. Chem. 271::12669-12673.
Pang, S.T. et al. 2002. . Gene expression profiling of androgen deficiency predicts a pathway of prostate apoptosis that involves genes related to oxidative stress. Endocrinology. 143::4897-4906.
Tusher, V.G., Tibshirani, R., and Chu, G. 2001. . Significance analysis of microarrays applied to the ionizing radiation response. Proc. Natl. Acad. Sci. U. S. A. 98::5116-5121.
Analysis of incidence of genital herpes in110pairs of couples.
YANG Xi，LINYa-zhen，XUYan-hui，et al.
（Zhanjiang Municipal Institute of Chronic Diseases，Zhanjiang524037，Guangdong，P.R.China）
摘要:目的 了解生殖器疱疹（GH）在夫妻间的发病情况，探讨近年来GH发病率不断升高的有关原因。 方法 对110例首诊为GH及其配偶110例的临床资料进行分析。 结果 夫妻同患GH73对（146例），占66.36%。其中男性首诊者47例，主要为复发性GH，女性首诊者26例，主要为亚临床感染及复发性GH。在73对患者中，夫妻一方首诊为GH后，有56例的配偶同时被诊为GH，其余17例在追踪2个月～1年后才被确诊。配偶被追踪诊断的GH中女性主要为亚临床感染，占51.11%（23/45），男性主要为复发性GH，占53.57%（15/28）。 结论 夫妻同患GH比例较高，亚临床感染发生率较高，皮肤呈多形性，极易导致误诊或漏诊，这些因素是GH易于传播的主要原因。
关键词:生殖器疱疹;接触追踪;发病率
近年来，GH的发病率在不断增长，与多种因素有关，其中主要原因是亚临床感染无客观临床表现，而以潜伏的形式存在，容易被忽视。同时是GH极易通过性接触传染，夫妻中一方患有GH，其配偶同患的可能性极大，却常常不能同时诊治，导致GH的继续传播。为了解GH在夫妻间的发病情况，现将首诊确诊为GH110例及其配偶的临床资料分析如下。
1 材料与方法
1.1 病例 来自2001年6月～2005年8月在我中心皮肤性病科门诊首诊被诊断为GH的已婚者110例及其能被追踪的110例配偶。
1.2 实验室检查 对有皮损的患者分别作HSV抗原及血清HSV-2抗体检查，对未发现皮损的首诊患者及其被追踪配偶除查血清HSV-2抗体外，取其近来出现过自觉症状的生殖器部位的皮肤粘膜组织液，男性尿道拭子、女性宫颈管拭子作HSV抗原检查。对部分患者作聚合酶链反应（PCR）检测，以对其结果进行对照。
1.3 诊断标准 根据生殖器疱疹的诊断标准，结合病史，临床表现及实验室检查进行综合分析。
1.4 临床分类 包括原发性:有非婚性接触或配偶感染史，首次发病，临床症状支持，HSV抗原检查阳性;复发性:病史及临床支持，多次发作，HSV抗原阳性，血清HSV-2抗体阳性或阴性;亚临床感染:病史支持。无客观临床表现，男性尿道，女性宫颈管拭子或有主观感觉的皮肤粘膜组织液HSV抗原检查阳性，血清HSV-2抗体阳性或阴性。
2 结果
2.1 发病情况 110例首诊为GH患者中，男71例，女39例。年龄21～63岁，年均30.5岁。男性及女性患者中分别有47例和26例的配偶同时患GH，即夫妻间同患GH73对，占66.36%。73对夫妻同患GH中，有56例的配偶同时被追踪诊断为GH，占76.71%，男性17例的配偶在追踪2个月～1年后被确诊，占23.29%。有37例首诊患者的配偶无同患GH，占33.64%。
2.2 病期 首诊与被追踪配偶所患GH的病期相似，原发性在5～30d;复发性1～4年不等;亚临床感染者病期未明。
2.3 临床分类与性别关系 见表1。
表1 73对夫妻同患及37例首诊不同患GH临床分类与性别情况（略）
2.4 临床表现 73对夫妻同患GH中，有31对夫妻均出现GH疑似皮损，占42.47%，7对均为亚临床感染，28对夫妻丈夫有皮损而妻子为亚临床感染，7对妻子有皮损而丈夫为亚临床感染者。在73对GH患者中，有典型皮损者46例，占31.51%，主要表现为生殖器部位呈集簇性或散在的小水泡。部位的发生率由高到低依次为男性的龟头、包皮、冠状沟及阴茎体;女性的大阴唇、小阴唇和阴蒂。有22例伴有腹股沟淋巴结肿大触痛，20例出现发热头痛、乏力等不适感。非典型皮损100例，占68.49%，主要表现为皮损多形性，其发生率依次为糜烂、溃疡、结痂、裂隙、红斑、丘疹、毛囊炎、疖肿和硬结。部位的发生率由高到低依次为男性的冠状沟、包皮、龟头、阴茎、尿道，阴囊、肛周;女性的宫颈、小阴唇、阴道、大阴唇、阴蒂、口腔及肛周。49例亚临床感染者有26例生殖器局部出现神经敏感症状，占53.06%。如阵发性灼热刺痛，放射性疼痛等。13例女性有白带异常，阴道不适或性接触时疼痛;10例男性尿道不适及排出清亮状分泌物。
2.5 非夫妻同患情况 有37对夫妻，仅一方患有GH，对另一方进行数月至近4年的追踪均未发现临床症状，同时定期做血清HSV-2抗体，男性尿道及女性宫颈管拭子HSV检查亦均为阴性。这些患者均在发病后立即避免与配偶密切接触或性接触时用安全套，但有6对夫妻，其中4例丈夫、2例妻子均为亚临床感染者，夫妻双方始终保持密切的性生活，而没有采取诸如使用安全套等防护措施，其配偶始终没有出现症状，血清HSV-2抗体一直阴性，PCR检测亦显示阴性。
2.6 婚外性接触史 73对夫妻同患GH中，夫妻双方均承认有婚外性接触史者12对，双方均承认有婚外性接触史者8对，其余53对夫妻中有一方承认有婚外性接触史，其中丈夫41例，主要为嫖娼占22例，其中为婚外性伴16例，同性恋者3例;妻子12例，均为婚外性伴。配偶无同患GH的37例中，承认有婚外性接触史者31例。
3 讨论
GH的传播已成为一个令人关注的公共卫生问题，椐WHO估计全球每年新发GH病例为2000万，居性传播性传染病（STD）的第四位，美国疾病防治与控制中心估计美国每年新发GH病例约30～50万 ［1］ 。而我国GH年均增长55.17% ［2］ ，排在规定疫报的8种性传播疾病的首位，并且易在夫妻间及性伴中造成传播。本研究显示，夫妻同患GH的比例较高，为66.36%，提示GH极易通过性接触传染，即夫妻一方患有GH，其配偶或性伴同患的可能性极大。所以任何一方患有GH，都应动员配偶及其性伴来检查，即使当时未发现GH也应继续追踪复查，定期做血清学检查。并对GH患者进行医学和性行为教育，鼓励其坚持正规使用安全套，有利于控制GH快速蔓延。
本文首诊发现病例男性比女性的比例高，分别占64.38%和36.62%，男女均以复发性GH为主，分别占68.09%和38.46%;而亚临床感染女性首诊和被追踪者分别为46.15%和51.11%，均明显高于男性的10.64%和32.14%，这与男性病情反复发作，症状较明显，部位较女性暴露，易引起注意而主动就诊有关。对于夫妻双方均为亚临床感染者，如果不是由于一方来诊治其他性病的同时做血清HSV-2抗体检查被发现，很可能长期被漏诊。
皮损的多形性和亚临床感染是GH难于诊断的主要原因，本文73对GH患者中，有典型皮损仅46例，占31.51%;非典型皮损100例，占68.49%，与邵氏 ［3］ 报道的55%和45%有较大差异。非典型皮损表现为多形性，如糜烂、溃疡、结痂、裂隙、红斑、丘疹、毛囊炎、疖肿和硬结等，致使临床上难于识别。因此有学者认为，对所有生殖器皮损，无论形态如何，均应考虑到生殖器疱疹的可能性 ［4］ 。此外，我们还发现在追踪的73例GH患者中，有6例（占8.22%）皮损位于口腔和肛周的外生殖器部位，追问病史均有口、肛—生殖器性交史。我们认为随着性行为方式的改变，对外生殖器部位的皮损亦不可忽视。关于亚临床感染，有研究显示70%～80%HSV-2生殖器疱疹感染临床上无症状 ［5，6］ ，且女性更为常见 ［5］ 。感染者中20%为有症状，60%有症状但患者或医生未能察觉或识别，另20%则为无症状的亚临床感染 ［5，6］ 。本文49例亚临床感染者中，有局部神经敏感症状者26例，占53.06%，女性有白带异常及阴道不适者占26.53%，男性有尿道不适及排出清亮状分泌物者占20.41%，与文献综述相似。这些患者往往是造成GH广泛传播的主要因素。
参考文献:
［1］唐晓青，杨秀莉，李春阳.生殖器疱疹的流行病学及预防与控制［J］.中国麻风皮肤病杂志，2002，18（3）:266～267.
［2］龚向东，叶顺章，张君炎，等.1991～2001年我国性病流行病学分析［J］.中华皮肤科杂志，2002，35（3）:178～182.
［3］邵椒青，胡逸琦，陈艳.荧光定量PCR检测疱疹病毒与临床分析［J］.中国麻风皮肤病杂志，2003，19（5）:511～512.
［4］赖伟红，邵长庚.生殖器疱疹的研究进展［J］.中国麻风皮肤病杂志，2002，18（4）:372～374.
［5］赖伟红，叶顺章，韩国柱.生殖器疱疹临床研究的某些新进展［J］.国外医学，皮肤性病学分册，2000，26（3）:162～166.
［6］邹勇莉，李谦和，徐延华.生殖器疱疹的诊断及治疗［J］.皮肤病与性病，2001，23（3）:16～18.
【摘要】 目的 胰岛素样生长因子（insulin-like growth factor,IGF）生成试验是临床常用的内分泌动态试验，常被视为评估GH不敏感综合征（GH insensitive syndrome,GHIS）矮小患者GH-IGF1生长轴功能技术之一。本文旨在观察：（1）IGF生成试验中的IGF1反应值（△IGF1）是否能够反映rhGH促生长效应；（2）IGF生成试验对GHD、ISS与GHIS三类患者是否具有临床鉴别价值。方法 对27例身材矮小患儿的IGF生成试验做一回顾性分析，平均年龄10.2岁（5.8~15.0岁），均处于青春发育期前期（Tanner Ⅰ期），其中原发性生长激素缺乏（GHD）15例、特发性矮小（ISS）11例和GH不敏感综合征（GHIS）即Laron综合征1例。IGF生成试验相关参数检测包括：血清GH峰值、IGF1和生长激素结合蛋白（GHBP）。依据生长速率（GV）探索△IGF1诊断敏感性和特异性的切点。结果 GHD与ISS患者血清IGF1SDS基础值和△%IGF1差异具有显著性（t=2.17，P=0.003；t=2.23,P=0.03）。GHD组患儿基础IGF1SDS与△%IGF1和△GV呈负相关（r=-0.79，P＝0.001及r=-0.59，P＝0.028）；GH峰值与△IGF1、△GV呈负相关（r=-0.78，P＝0.001及r=-0.64，P＝0.01）；△%IGF1与△GV呈正相关（r=0.63，P＝0.015）。ISS组患者△IGF1与HtSDS及IGF1基础值呈负相关（r=-0.61，P＝0.047；r=-0.64，P＝0.036）；由ROC曲线可见△IGF1对ISS患儿判断GH疗效具有较高敏感性和特异性的切点约为165.9μg/L。结论 （1）IGF生成试验是完全性GHI可靠的实验诊断；（2）IGF生成试验具有一定的预测rhGH促生长效应的作用；（3）本文未能对部分性GHI提供临床诊断信息。
【关键词】 生长调节素类；生成试验；分析
Insulin-like growth factor generation test and its clinical value
WANG Xiu-min,WANG Wei,DU Xiao-fei,et al.Department of Pediatrics,Ruijin Hospital,The Second Medical University of Shanghai,Shanghai 200000,China
【Abstract】 Objective The insulin-like growth factor（IGF）generation test has been used to assess growth hormone（GH）responsiveness in children with short stature in clinic.The aim of this study was to seek a cutting value （1） in effect of prediction for growth promoting with recombinant human GH（rhGH） therapy in children with idiopathic short stature（ISS） and （2） in differential diagnosis between GH deficiency （GHD） ISS and GH insensitive syndrome（GHIS）.Methods Twenty-seven pre-pubertal children with short stature（including 15 GHD,11 ISS and 1 GHIS）,mean age 10.2 years（5.8~15.0）,were retrospectively studied.All the patients received rhGH treatment over one year except the GHIS patient.Serum GH peak value,IGF1 and GH binding protein（GHBP） levels were measured.The diagnostic cut-off line of IGF1 responding levels（△IGF1）during standard IGF generation test was sought on the basis of growth velocity（GV）.Results Among the GHD and ISS groups,distinct differences were shown in serum basal IGF1SDS levels（t=2.17，P=0.003）and △%IGF1 levels（t=2.23，P=0.03）.In GHD group we found negative relationships between the basal IGF1SDS and △%IGF1（r=-0.79，P＝0.001）,between the basal IGF1SDS and △GV（r=-0.59，P＝0.028）,between GH peak level and the △IGF1 level（r=-0.78,P=0.001）and between GH peak level and △GV（r=-0.64,P=0.01）.Positive relationship was shown between △%IGF1 and △GV（r=0.63，P＝0.015）in GHD patients.In ISS group,negative relationships were shown between △IGF1 and HtSDS（r=-0.61，P＝0.047）and between △IGF1 and basal IGF1 value （r=-0.64，P＝0.036）.The ROC plot showed that the best cut-off line of △IGF1 for prediction of rhGH therapeutic effect was likely 165.9μg/L.Conclusion （1） The IGF generation test is a reliable clinical laboratory diagnostic parameter for complete GHI;（2） IGF generation test was suspected valuable in prediction of growth promoting effect in short stature with rhGH treatment;（3） The diagnostic value of IGF generaton test for partial GH insensitivity need to be clarified.
【Key words】 somatomedins;diagnosis;growth hormone insensitive syndrome
胰岛素样生长因子（insulin-like growth factor,IGF）生成试验是临床常用的内分泌动态试验，常被视为评估身材矮小者GH-IGF1生长轴功能技术之一，如GH不敏感综合征（GH insensitive syndrome,GHIS）即Laron综合征。近年来，随着研究下丘脑-垂体生长轴病理缺陷机制及重组人生长激素（rhGH）临床应用的不断深入，人们对IGF生成试验有了新的认识，但就其临床应用价值仍存有一些疑惑。本文对27例身材矮小患儿的IGF生成试验做一回顾性分析，旨在观察:（1）IGF生成试验是否能够预测rhGH促生长疗效；（2）IGF生成试验对原发性生长激素缺乏（GHD）、特发性矮小（ISS）及GHIS患者是否具有临床鉴别价值。
1 资料与方法
1.1 一般资料 27例身材矮小者均为我院儿童生长发育中心门诊就诊患儿，男18例，女9例；平均年龄10.2岁（5.8~15.0岁），均处于青春发育期前期（Tanner Ⅰ期）。按下述入选标准分为GHD、ISS和GHIS三组，其中GHD组15例，ISS组11例和GHIS 1例，见表1。GHD及ISS患者皆于试验后接受rhGH治疗，剂量0.1u/（kg·d），临睡前皮下注射，并随访治疗1年后的生长速率（GV）。
GHD临床诊断依据：（1）身高低于正常平均水平-2SD；（2）生长速率明显降低；（3）骨龄（BA）落后实际年龄2岁以上；（4）GH药物激发试验血清GH峰值＜5μg/L；（5）无特异性内分泌功能紊乱及慢性器质性疾病和心理异常。ISS临床诊断依据：（1）身高低于正常平均水平-2SD；（2）生长速率正常或降低；（3）出生体重正常；（4）GH药物激发试验血清GH峰值＞10μg/L；（5）无其他内分泌遗传代谢紊乱及慢性器质性疾病和心理异常。GHIS临床诊断依据：（1）身高低于正常平均水平-2SD；（2）血清GH峰值明显增高（多＞20μg/L）；（3）BA明显延迟；（4）血清IGF1、胰岛素样生长因子结合蛋白3（IGFBP3）及生长激素结合蛋白（GHBP）明显低下；（5）rhGH治疗无效。
1.2 方法
1.2.1 GH激发试验 所有患儿在初诊时均接受两项GH药物激发试验（可乐宁、精氨酸）。血清GH检测采用放射免疫定量药盒（德普公司产品），药盒敏感度0.9ng/ml，组内和组间CV分别为>5%和>7%。
1.2.2 IGF1生成试验 采用rhGH皮下注射法，剂量为0.1u/（kg·d），连续4或7日临睡前注射，在试验前及试验结束后次日晨分别采血测定血清IGF1及GHBP。IGF1采用放免检测技术，药盒为法国Immunotech公司产品，敏感度为3ng/ml，组内和组间CV为7.4%和15.5%；GHBP采用酶联免疫检测技术（ELISA），药盒为美国DSL公司产品，敏感度为1.69pmol/L，组内和组间CV为3.17%和6.2%。
1.2.3 IGF生成试验诊断参数计算 依据GH治疗1年后的GV变化，计算血清△IGF1诊断敏感性和特异性的计算和绘制受试者工作特性曲线（receiver operating characteristic curve,ROC曲线）。敏感性（预测疗效佳组中真阳性比例）=疗效佳组中真阳性病例数/（真阳性例数+假阴性例数），特异性（疗效欠佳组真阴性比例）=疗效欠佳组中真阴性病例数/（真阴性例数+假阳性例数），根据ROC确定临床对ISS患儿判断GH疗效具有较高敏感性和特异性的切点。
1.3 统计学方法 分析资料均以均数±标准差（±s）表示，组间相关指标采用t检验和方差分析（ANOVA，两两比较用最小极差法，即LSD检验），P<0.05为差异有显著性。所有统计分析全部由SPSS 11.0软件完成。
表1 两组身材矮小患儿一般资料 （略）
注：△IGF1、△IGFBP3分别表示激发后IGF1和IGFBP3与激发前相比的差值，△%IGF1、△% IGFBP3分别表示激发后IGF1和IGFBP3与激发前相比差值的百分数，GV为生长速率，△GV为rhGH治疗后GV增加值，△HtSDS为rhGH治疗后身高SD积分的增加值
2 结果
2.1 IGF生成试验基础值 GHD组平均血清IGF1SDS基础值为-1.5±0.8，ISS组为-0.90±0.3，两组差异具有非常显著性（t=2.17，P=0.003）。根据ROC曲线分析，可见判别GHD与ISS的IGF1截断值为221.6ng/ml（见图1、图2），其敏感性为54.5%，特异性为84.6%。GHD组平均血清IGFBP3基础值为（4352.85±369.77）μg/L，GHBP为（115.0±11.9）pmol/L；ISS组IGFBP3为（6186.444±433.79）μg/L、GHBP为（148.0±22.0）pmol/L，两组差异均无显著性（IGFBP3 t=1.54，P=0.14及GHBP t=1.45，P=0.16），见表1。
图1 患者GH激发峰值对应IGF1基础值分布图 （略）
图2 患者GH峰值、IGF基础值和IGF基础值 （略）
2.2 IGF生成试验反应值 GHD与ISS患者血清IGF1激发反应差值百分率（△%IGF1）差异具有显著性［（51.9±10）%与（38.7±7）%，t=2.23，P=0.03）］；GHD与ISS患者血清IGFBP3SDS反应值差异亦具有显著性（1.17±1.0与4.14±.72；t=3.17，P=0.005）（表1）。血清GHBP反应值未见显著性差异［（151.0±12.7）与（168.5±22.5）pmol/L；t=1.57，P=0.13）］。提示IGF生成试验检测GHBP反应值参数无观察意义。
2.3 相关性分析
2.3.1 GHD组 基础IGF1SDS与△%IGF1和△GV呈负相关（r=-0.79，P＝0.001及r=-0.59，P＝0.028）；GH峰值与△%IGF1、△GV呈负相关（r=-0.78，P＝0.001及r=-0.64，P＝0.01）；IGF1反应值与rhGH治疗后1年血清IGF1呈正相关（r=0.89，P＝0.07）；△%IGF1与△GV呈正相关（r=0.63，P＝0.015）；△%IGF1与△%IGFBP3呈正相关（r=0.727，P＝0.011）。可见GHD患者IGF1基础值与IGF生成试验的反应程度及rhGH替代治疗疗效之间存在一定关联。
2.3.2 ISS组 患者△%IGF1与HtSDS及IGF1基础值呈负相关（r=-0.61，P＝0.047；r=-0.64，P＝0.036）。△IGF1对应rhGH治疗前后的GV变化见图2。
2.4 预测rhGH疗效分析 本文以血清△IGF1作为判断预测GH疗效（GV）切点，分别计算疗效佳组中真阳性和假阴性病例数及疗效欠佳组中真阴性和假阳性病例数，并分别计算出该切点判断疗效的敏感性和特异性。可见△IGF1对ISS患儿判断GH疗效具有较高敏感性和特异性的切点约为165.9μg/L（见表2、图3）。
表2 IGF生成试验中GV变化判断的ROC点分析△IGF1（略）
图3 ISS患者IGF生成试验△IGF1值对应rhGH治疗前和治疗1年后的GV变化 （略）
3 讨论
临床设计应用IGF1生成试验至今已有20余年，其试验原理是通过外源性GH刺激外周靶细胞分泌IGF，以评价GH的刺激效应。故其检测目的应包括两方面：（1）检测GH受体（GHR）对其配体反应的敏感性：当GHR功能活性正常时，外源性GH刺激后血清IGF1明显提高；反之，GHR功能缺陷则IGF1反应无明显变化，由此可诊断GHR不敏感（GHI）及预测外源性GH促生长疗效。（2）检测内源性GH功能活性：当内源性GH分泌数量正常而功能缺陷时，采用外源性GH刺激能产生良好应答反应，IGF较基础值明显提高。
基于上述观点，IGF生成试验曾设计作为GH-IGF轴动态功能试验被用于识别完全性GH不敏感（GHI，即Laron综合征）。1994年Blum等［1］提出该试验的实验诊断标准（积分系统），即在标准注射外源性GH持续4天后，血清IGF1反应值较基础值升高<15μg/L、IGFBP3＜0.4mg/L。本文观察对象均未达到上述实验诊断标准，结合血清GHBP值正常及临床良好的rhGH促生长疗效，故排除完全性GHI。对照我院1例可疑GHI患儿，其IGF生成试验血清IGF1基础值为19.1μg/L，反应值10.0μg/L；IGFBP3基础值663.1μg/L，反应值352.1μg/L；GHBP 15pmol/L及无rhGH治疗，符合完全性GHI（另文报道）。可见IGF生成试验对鉴别完全性GHI具有一定临床价值。
有关IGF生成试验对生长激素缺乏（GHD）、非GHD的特发性矮小（ISS）及部分性GHI的鉴别诊断意义已有不少观察报道，但结果大相径庭，其主要问题可能还在于不同病理缺陷的检测结果过分重叠。目前尚未建立一致的临床实验鉴别标准。已有报道单独依据GH激发峰值鉴别GHI无实用价值。1995年Attie等提出［2］：20%ISS患者呈现血清GHBP和IGF1低下，提示可能存在对GH作用抵抗，但IGF生成试验未达到完全性GHI实验诊断标准，即部分性GHI可能成为ISS的一种病因。同年Goddard等又首次报道［3］在ISS患者中存在GH受体基因突变，是部分性GHI的分子病理机制。故提示该激发试验在一定程度上能识别部分性GHI，预示rhGH疗效不良，或提示GH治疗应加大剂量。本组临床观察显示，GHD与ISS患者中血清IGF1基础值差异具有显著性；在ISS组中仅提示△IGF1为165μg/L时可能GH临床疗效较佳，但可能限于观察ISS样本有限，未显示有统计价值的识别部分性GHI的诊断截断标准。
至于IGF生成试验在ISS患儿中临床应用价值仍有一定的争议［4］，究其原因可能与缺乏统一的检测程序及与试验结果判断标准有关，如GH给药剂量和时间、采血时间、IGF等测定方法，以及缺乏不同年龄、性别对外源性GH反应评价的正常参考标准等。目前一些学者已提出［5~8］，外源性GH刺激剂量对试验结果至关重要，大剂量对试验敏感性无明显优势，宜采用小剂量刺激法，以增加试验检测反应的敏感性，尤其是识别部分性GHI；此外，适当延长刺激时间可能在临床鉴别诊断中有助于改善试验结果的重叠现象，检测血清IGF1反应的同时可检测IGFBP3，两者结合可以获得更大信息。
总之，IGF生成试验的临床应用价值正在不断被认识和论证。本文观察结果显示：（1）IGF生成试验是完全性GHI可靠的实验诊断依据；（2）IGF生成试验具有一定的预测rhGH促生长疗效；（3）在ISS患者中对部分性GHI提供临床鉴别诊断信息还有待于不断扩大观察样本数量，以充实可靠的相关信息。
【参考文献】
1 Rosenfeld RG，Albertsson-Wikland K,Cassorla F,et al.Diagnostic controversy:the diagnosis of childhood growth hormone deficiency revised.J Clin Endocrinol Metab，1995，80:1532-1540.
2 Blair JC,Camacho-Hubner C.Standard and low-dose IGF1 generation tests and spontaneous growth hormone secretion in children with idiopathic short stature.Clinical Endocrinol,2004,60（2）:163-168.
3 Attie KM,Carlsson LM,Rundle AC,et al.Evidence for partial growth hormone insensitivity among patients with idiopathic short stature.The national Cooperative Growth Study.J Pediatr,1995,127:244-250.
4 Cotteril AM,Camacho-Hubner C,Woods,et al.The insulin-like growth factor 1 generation test in the investigation of short stature.Acta Paediatr.1994,399:128-130.
5 Goddard AD,Dowd P,Chernausek S,et al.Partial growth-hormone insensitivity:the role of growth-hormone receptor mutations in idiopathic stature.J Paediatr,1997,131:51-55.
6 Buckway CK,Guevara-Aguirre J,Pratt KL,et al.The IGF-1 generation test revisted:a marker of GH sensitivity.J Clin Endocrinol Metab,2001,86:5176-5183.
7 Buckway CK,Selva KA,Pratt KL,et al.Insulin like growth factor binding protein-3 generation as a measure of GH sensitivity.J Clin Endocrinol Metab,2002,87:4754-4765.
8 Darendelilre F,Ocal C,Bas F,et al.Evaluation of insulin like growth factor（IGF）-1 and IGF binding protein-3 generation test in short stature.J Clin Endocrinol Metab,2005,18:443-452.
作者单位： 200000 上海，上海第二医科大学附属瑞金医院儿内科
（编辑：乔 晓）
【摘要】 生长激素（GH）在正常的心血管生理中发挥着重大的作用。不论是GH过量还是GH分泌不足，均可能导致心血管疾病的发生。老年人GH的相对不足也可增加心血管疾病的发病率。小剂量的GH补充疗法，可用于治疗充血性心力衰竭（CHF）及保持老年人的健康。GH释放激素（GHRH）的初步临床研究显示可减轻GH疗法的副作用。
【关键词】 生长激素；心血管疾病；综述文献
生长激素（growth hormone,GH）控制着身高的增长，同时还有许多其他功能，如参与营养物质的代谢、骨髓肌的功能和身体组成成分等的调节，甚至心理健康的维持。GH在心血管生理中也起着重要的作用，对心脏的发育以及成年后心脏正常形态及功能的维持均具有重要的影响。GH分泌过多或不足均可导致心血管疾病。近年来，有关GH与心血管疾病的关系的研究取得了一些进展，现综述如下。
1 GH对心血管系统的生理和病理作用
GH可使健康人的心输出量增加，心率加快，心肌收缩力增强，收缩末期容积减少，而对舒张功能影响不大。在动物模型中对GH产生这种作用的机制进行了研究。GH对心脏的作用往往经由心脏局部产生的胰岛素样生长因子（IGF）介导，后者增强大鼠心肌细胞收缩力是通过刺激Ca2+从终末池和有纤维膜下池中释放的结果。Stromer等［1］注意到大鼠心肌在GH作用下对Ca2+的反应性增强。Ito等［2］发现IGF-1可使大鼠心肌细胞的肌球蛋白（myosin）和肌钙蛋白1的基因转录增强，因而使肌纤维增粗、收缩力增强。Cittadini等［3］对垂体性侏儒大鼠的研究发现，尽管该大鼠对异丙肾上腺素的反应性降低，但是β-受体的密度、亲和力等都没有改变。用GH治疗后，心功能得到改善，与心得安合用时疗效也没有改变。说明GH改善心功能的作用与β-受体路径无关。GH在心血管生理和病理中的重要性可以从GH分泌不足和GH分泌过量的病人中得到充分的体现。
1.1 GH分泌不足（GHD） 许多年前即已认识到，垂体切除者或患垂体性侏儒症的病人，其心血管功能发生紊乱的危险性明显增加。在瑞典对333例GHD病人的回顾性研究中，其心血管疾病的发病率和死亡率均显著增加［4］。GHD者血清甘油三酯和总胆固醇水平升高，高密度脂蛋白胆固醇（HDL）下降。这种脂质代谢紊乱增加了心血管疾病发生的危险性。GHD者心脏功能的改变表面为“低动力学型”，即心肌收缩力减弱，心输出量减少，工作效率下降。一般认为其心脏功能和结构的受损程度与GHD发生的早晚有关，先天性GHD表现得较为严重。但最近的研究否定了这种观点，认为幼年时或成年后患GHD者其左心功能减退的程度没有明显差异［5］。
大量的临床和动物实验证明，对GHD者用GH替代治疗可以改善心脏的功能。Caidahl等［6］对GHD病人用小剂量GH 0.5u/（kg·w）治疗6个月，病人的心血管功能明显改善，心输出量和左室重量增加，外周血管阻力下降，而对左室舒张功能和血压等没有不利影响。Jahannson等［7］研究7例GHD病人，经长达3~4年的GH治疗后发现心脏指数有显著改善，最大运动负荷量在治疗6个月后即恢复正常，治疗42个月后甚至高于正常。有学者用Hexarelin（一种新近合成的能促进GH释放的六肽）治疗因用抗GH释放激素抗体（GHRH-Ab）形成GHD的大鼠，发现可减轻心肌缺血再灌注性损伤，该大鼠冠状动脉对血管紧张素Ⅱ的敏感性增加和血管内皮合成前列环素减少，经用Hexarelin可以逆转。说明GH对维持血管内皮的正常功能也具有重要作用［8］。Fazio等［9］研究了25例先天性GHD病人，发现其中12例经用rGH治疗12个月后，室壁厚度增加，张力作用有所减弱，心肌收缩功能和心输出量显著改善，认为GH在心室壁厚度和张力的调节中具有重要的作用，其机制为影响心肌组织的生长。
GHD病人血中纤维蛋白原和纤溶酶原激活剂抑制因子（PAI）水平增高，血清胆固醇、低密度脂蛋白胆固醇（LDL）和甘油三酯水平也增高。这些因素与动脉粥样硬化疾病密切相关。而经过GH治疗后其增高的血脂得以降低，理论上患心血管疾病的危险性相应下降。Cuneo等［10］对GHD病人用GH治疗6个月后，总胆固醇和LDL明显下降，而HDL则没有变化。小剂量的GH可使胆固醇水平下降至相当于用辛伐他丁治疗3个月的水平。
但也有报道GH可使载脂蛋白a［Lp（a）］增高，而Lp（a）被认为是心血管疾病的独立危险因素。如Oscarsson等［11］给8例中年超重者连续皮下注射GH 14天，发现血清Lp（a）增加了42%，而LDL则有所下降。Laron等［12］研究发现给3组病人（7例患GHD的年轻人，7例患Turner综合征的女孩，15例肾衰竭患儿）用GH治疗1个月后，Lp（a）增加了67~100%，有些病人其Lp（a）达到了可以导致心血管疾病的危险水平（300mg/L）。而用IGF-1治疗则可使Lp（a）水平下降。
1.2 GH分泌过量 GH分泌过量是肢端肥大症的特征。患者常并发血管疾病，其死亡率增高3~4倍。大量研究证实GH过量可对心血管产生许多不利影响。
1.2.1 临床研究 临床观察发现心脏对过量GH引起的压力和容量的超负荷状态能够产生适应，主要表现为向心性肥厚。这种心脏肥厚不同于单纯性的向心性肥厚，它不引起心室内径的变化，除非到了晚期出现心力衰竭。因此其室内压和室壁张力变化不大。在长期GH高分泌状态下，心脏呈现双心室的向心性肥厚。双心室舒张期充盈均受限，收缩功能在休息时变化不大，但当运动时，左室的射血分数没有相应增加，说明心功能储备受损。肢端肥大症疾病的早期，心脏收缩力增强，心输出量增加，外周血管阻力下降，呈现高动力学状态，心脏尚未出现形态学的改变。随着病程进展，出现心肌肥厚和间质增生，心室的舒张功能减弱，周围血管灌注减少，心输出量开始下降，可出现劳力性呼吸困难。到严重阶段，心室出现扩张，心输出量显著减少，外周血管阻力增加，有时会出现高血压［13］。此时组织病理学检查可见心肌组织广泛纤维化，部分心肌坏死，伴淋巴细胞和单核细胞浸润。
心脏对GH高分泌的反应和病理改变部分是可逆的。生长抑素的同类物奥曲肽（Octreotide）即可逆转肢端肥大症的心脏肥厚病变。奥曲肽可以抑制GH的释放，制止GH的脉冲式分泌。Lim等［14］对肢端肥大症病人用奥曲肽治疗1周，肥厚心室的重量即明显减轻，用药2个月后继续减轻，但没有恢复到正常水平。
1.2.2 动物研究 用外科手术将大量分泌GH的肿瘤（GH3细胞系）移植到大鼠身上，发现心脏重量增加138%；肝、肾、脾、肾上腺和总体重均有所增加，但未达到心脏重量增加的程度；同时，骨骼肌和心肌的DNA合成增加。去除肿瘤后，各器官出现萎缩。Timsit等［15］对携带GH分泌瘤的大鼠的分子研究显示肌球蛋白基因的表达改变，心脏V3肌球蛋白异构体（isomyosin）浓度相对于V1肌球蛋白异构体增加。在V3含量丰富的心脏观察到存在正常的和超正常的强力收缩，而缩短速度则正常。肌动蛋白与肌球蛋白之间的横桥数量增加，对钙的敏感性也增加，使心脏收缩的效率提高。心脏分子特性的内在变化可能有助于心脏在过量的GH环境中保持其功能。
2 GH在心血管疾病防治方面的应用
2.1 充血性心力衰竭 初步的研究表明，不论是GH水平正常还是GH分泌不足的CHF病人，短期应用GH治疗均可改善心功能，但也有相反的报道。目前尚没有大规模的GH治疗CHF的研究。
2.1.1 支持性的研究报告 主要集中在对原发性扩张性心肌病心力衰竭的治疗。Fazio等［16］对7例患轻至重度心力衰竭的原发性心肌病病人用GH每周14u，治疗3个月，发现心肌重量显著增加，左室内径缩小，心输出量增加，肺动脉压下降，冠脉血流量也增加，心肌能量代谢改善，临床症状明显好转。Vdterrani等［17］对12例CHF病人应用rGH连续静脉输注24h，平均心脏指数显著增加（从2.1升到3.3,P＜0.01），肺动脉压下降25%，说明短期应用GH可改善心功能。另有2例终末期心力衰竭病人在其他常规治疗的基础上加用GH也同样使心功能明显改善［18］。Capaldo等［19］研究发现GH可以减少原发性扩张性心肌病心肌组织去甲肾上腺素的含量，并使循环血中醛固酮的水平下降，因而认为GH可以作为心力衰竭治疗的待选药物之一。有学者测定了9例CHF病（7例为原发性扩张性心肌病，2例为冠心病）的GH和IGF-1水平，发现较正常人为低［20］。另12例严重扩张性心肌病病人则表现为夜间GH的自然分泌不足，并且与射血分数相关［21］，因此GHD可能在心肌病的发病机制中起一定的作用。
GH可以改善CHF的心功能的支持性证据主要还是来自动物研究。如Yang等［22］在给大鼠心脏结扎冠状动脉或假手术后4周（所有经冠状动脉结扎的大鼠于1周后心电图均出现Q波和心肌梗死图形），给予GH治疗15天，结果用GH治疗者与未用GH者相比较，治疗组的心肌收缩力增强，心输出量增加，左室舒张末期压和外周阻力下降，心率保持不变。另一个研究对诱导CHF的大鼠在用GH治疗前，先用卡托普利治疗3个月。结果表明接受两种治疗的大鼠的心功能均得到改善，并用两药合用比单用一种药物心功能改善更为显著。早期应用于GH治疗还可以减轻实验性心肌梗死的心室重构，并显著改善心功能，而且在改善心功能的同时不引起心室肥厚［23］。尤其引人注目的是还使大面积心肌梗死大鼠的心脏指数增加22%［24］。
2.1.2 不支持的证据 有研究发现GH仅使扩张性心肌病病人左室重量增加，而心肌功能未见改善。将50例扩张性心肌病病人随机分为两组，一组用人重组GH（rhGH）2IU/d治疗12周，另一组用安慰剂作对照（心力衰竭的常规治疗照常进行）。结果rhGH组的左心室重量较对照组增加27%（P=0.0001），但两组的左室收缩期室壁张力、动脉平均压、全身血管阻力却未见明显差异，NYHA心功能分级、左室射血分数和6min走路距离等心功能指标也没有明显改善，且有1例因心力衰竭加重而退出试验［25］。Shen等［26］在对用快速心室起搏而诱发心力衰竭狗的研究中也得到类似的结果，但左室收缩和舒张功能及外周血管阻力却没有显著变化。
2.2 心肌梗死后室壁瘤的预防 研究证明［27］GH对预防心肌梗死后室壁瘤的形成有效。对诱发心肌梗死的大鼠给予GH，电子显微镜检查显示非GH治疗组大鼠心脏的心肌纤维在72h内完全散乱，而治疗组散乱现象明显减轻；治疗组的室壁瘤发生率为10%，而非治疗组则为32%。另有研究观察心肌梗死大鼠给予GH或GH加β-受体阻滞剂治疗的效果，与对照组相比，GH组大鼠的室壁瘤形成减少，而GH加β-受体阻滞剂组的室壁瘤形成反而增加。
2.3 老年人心血管疾病的防治 给予老年人GH补充治疗可能是减少其心血管疾病的相关危险因素的有效措施。有研究［28］对26例正常男性（年龄61~81岁）给予GH 0.03mg/kg，每周3次，治疗6个月，发现脂肪组织显著减少（14%），身体构成成分得到改善。对健康老年妇女用rGH 0.025mg/（kg·d）治疗4周后，也出现类似的有益的变化［29］。研究显示GH补充疗法可使全身脂肪组织减少，腰围/臀围比例下降，并可减少其他一些危险因素，所以GH可能对降低老年人的心血管疾病发病率和病死率有效。但是应该指出的是，不是所有老年人均存在GH分泌不足。而且GH补充法也有一些副作用，如部分人出现血糖和收缩压升高［28］。同时，由于个体对GH的敏感性不同，所以很难确定对每个病人均合适的所谓标准剂量。有关长期使用GH可能出现的副作用和不良反应尚待进一步研究。
有人用GHRH治疗老年人中GH和IGF-1水平下降者。Corpas等发现给老年人皮下注射GHRH 2次/d，可使GH和IGF-1水平升高，另外，GHRH还可以刺激GH的夜间脉冲式分泌。反复给老年人静脉注射GHRH可以恢复其已受到抑制的GH对GHRH的反应性。GHRH与GH相比有一些优点。GHRH为一种小分子，将来可能提供经口服和（或）经鼻腔和皮肤给药的剂型。在使用GHRH时，可用对抗激素加以调节，这样可以减少不良反应，如减轻对血压、脉搏、体温或血糖的影响。故有可能成为可供选择的GH替代疗法之一。
【参考文献】
1 Stromer H,Cittadini A,Douglas PS,et al.Exogenously administered growth hormone and insulin-like growth factor-I alter intracellular Ca2+ handling and enhance cardiac performance.In vitro evaluation in the isolated isovolumic buffer-perfused rat heart.Circ Res，1996,79（2）:227-236.
2 Ito H,Hiroe M,Hirata Y,et al.Insulin-like growth factor-I induces hypertrophy with enhanced expression of muscle specific genes in cultured rat cardiomyocytes.Circulation，1993，87（5）:1715-1721.
3 Cittadini A,Stromer H,Vatner DE,et al.nsequences of growth hormone deficiency on cardiac structure,function,and beta-adrenergic pathway：studies in mutant dwarf rats.Endocrinology，1997，138（12）:5161-5169.
4 Rosen T,Bengtsson BA.Premature mortality due to cardiovascular disease in hypopituitarism.Lancet，1990，336（8710）:285-288.
5 Longobardi S,Cuocolo A,Merola B,et al.Left ventricular function in young adults with childhood and adulthood onset growth hormone deficiency.Clin Endocrinol （Oxf），1998，48（2）:137-143.
6 Caidahl K,Eden S,Bengtsson BA.Cardiovascular and renal effects of growth hormone.Clin Endocrinol （Oxf），1994,40（3）:393-400.
7 Johannsson G,Bengtsson BA,Andersson B,et al.Long-term cardiovascular effects of growth hormone treatment in GH-deficient adults.Preliminary data in a small group of patients.Clin Endocrinol （Oxf），1996,45（3）:305-314.
8 De Gennaro Colonna V,Rossoni G,Bernareggi M,et al.Hexarelin,a growth hormone-releasing peptide,discloses protectant activity against cardiovascular damage in rats with isolated growth hormone deficiency.Cardiologia，1997,42（11）:1165-1172.
9 Fazio S,Cittadini A,Sabatini D,Merola B,et al.Growth hormone and heart performance.A novel mechanism of cardiac wall stress regulation in humans.Eur Heart J，1997，18（2）:340-347.
10 Cuneo RC,Salomon F,Watts GF,et al.Growth hormone treatment improves serum lipids and lipoproteins in adults with growth hormone deficiency.Metabolism，1993,42（12）:1519-1523.
11 Oscarsson J,Ottosson M,Wiklund O,et al.Low dose continuously infused growth hormone results in increased lipoprotein（a） and decreased low density lipoprotein cholesterol concentrations in middle-aged men.Clin Endocrinol （Oxf），1994，41（1）:109-1016.
12 Laron Z,Wang XL,Klinger B,et al.Growth hormone increases and insulin-like growth factor-I decreases circulating lipoprotein（a）.Eur J Endocrinol，1997，136（4）:377-381.
13 Fazio S,Cittadini A,Cuocolo A,et al.Impaired cardiac performance is a distinct feature of uncomplicated acromegaly.J Clin Endocrinol Metab，1994，79（2）:441-446.
14 Lim MJ,Barkan AL,Buda AJ.Rapid reduction of left ventricular hypertrophy in acromegaly after suppression of growth hormone hypersecretion.Ann Intern Med，1992,117（9）:719-726.
15 Timsit J,Riou B,Bertherat J,et al.Effects of chronic growth hormone hypersecretion on intrinsic contractility,energetics,isomyosin pattern,and myosin adenosine triphosphatase activity of rat left ventricle.J Clin Invest，1990，86（2）:507-515.
16 Fazio S,Sabatini D,Capaldo B,et al.A preliminary study of growth hormone in the treatment of dilated cardiomyopathy.N Engl J Med.1996，334（13）:809-14.
17 Volterrani M,Desenzani P,Lorusso R,et al.Haemodynamic effects of intravenous growth hormone in congestive heart failure.Lancet，1997，349（9058）:1067-1068.
18 ODriscoll JG,Green DJ,Ireland M,et al.Treatment of end-stage cardiac failure with growth hormone.Lancet，1997，349（9058）:1068.
19 Capaldo B,Lembo G,Rendina V,et al.Sympathetic deactivation by growth hormone treatment in patients with dilated cardiomyopathy.Eur Heart J，1998，19（4）:623-627.
20 Acevedo M,Corbalan R,Godoy I,et al.Growth hormone deficiency in patients with chronic heart failure。Rev Med Chil，1997，125（1）:30-35.
21 Giustina A,Lorusso R,Borghetti V,et al.Impaired spontaneous growth hormone secretion in severe dialated cardiomyopathy.Am Heart J，1996，131（3）:620-622.
22 Yang R,Bunting S,Gillett N,et al.Growth hormone improves cardiac performance in experimental heart failure.Circulation，1995，92（2）:262-267.
23 Cittadini A,Grossman JD,Napoli R,et al.Growth hormone attenuates early left ventricular remodeling and improves cardiac function in rats with large myocardial infarction.J Am Coll Cardiol，1997,29（5）:1109-1116.
24 Duerr RL,McKirnan MD,Gim RD,et al.Cardiovascular effects of insulin-like growth factor-1 and growth hormone in chronic left ventricular failure in the rat.Circulation，1996,93（12）:2188-2196.
25 Osterziel KJ,Strohm O,Schuler J,et al.Randomised,double-blind,placebo-controlled trial of human recombinant growth hormone in patients with chronic heart failure due to dilated cardiomyopathy.Lancet，1998,351（9111）:1233-1237.
26 Shen YT,Woltmann RF,Appleby S,et al.Lack of beneficial effects of growth hormone treatment in conscious dogs during development of heart failure.Am J Physiol，1998，274（2 Pt 2）:456-466.
27 Castagnino HE,Toranzos FA,Milei J,et al.Preservation of the myocardial collagen framework by human growth hormone in experimental infarctions and reduction in the incidence of ventricular aneurysms.Int J Cardiol，1992,35（1）:101-114.
28 Rudman D,Feller AG,Nagraj HS,et al.Effects of human growth hormone in men over 60 years old.N Engl J Med，1990，323（1）:1-6.
29 Thompson JL,Butterfield GE,Marcus R,et al.The effects of recombinant human insulin-like growth factor-I and growth hormone on body composition in elderly women.J Clin Endocrinol Metab，1995,80（6）:1845-1852.
作者单位： 710089 陕西西安，解放军第141医院
（编辑：黄鉴一）
生殖器疱疹（GH）是由单纯疱疹病毒（HSV-Ⅱ）感染引起的一种常见的性传播疾病（STD），临床表现变化多样。典型的临床症状出现在生殖器部位，女性皮损位于外阴、肛周及臀部，约有90%同时侵犯宫颈，损害为水疱、脓疱、溃疡、结痂；疑似临床表现为生殖器部位的细小的线状溃疡、裂隙、硬结，非特异发生红斑及宫颈糜烂等。笔者在2001～2004年，对上述皮损30例女性患者进行HSV-Ⅱ DNA检测，现将结果报告如下。
1 资料与方法
1.1 一般资料 30例均为女性，年龄20～45岁，病程2天～1年。9例有婚外性生活史，6例认为配偶有冶游史，18例为宫颈糜烂Ⅱ度，患者自觉分泌物增多，部分伴腰部不适或腰酸，7例性生活后分泌物伴血丝，7例表现为裂隙，皮损均位于近大阴唇与侧腹股沟交界处，长约0.5～1cm，其中3例性生活后自觉外阴局部有破损，并伴刺痛，4例无明显自觉症状，3例表现为硬结，绿豆大小，呈正常肤色或浅褐色，1例位于会阴上部，2例位于大阴唇均伴自觉局部瘙痒，尤以月经前后明显。1例表现为尿道口红肿，伴有明显尿道刺激症状。
1.2 实验室检查 取皮损、分泌物行PCR检测HSV-Ⅱ DNA，并根据患者临床症状做相应检查，对排除其他STD感染。
2 结果
见表1。HSV-Ⅱ DNA检测阳性中有7例合并其他STD，其中1例合并早期梅毒，5例合并NSGⅠ，1例合并尖锐湿疣（CA）和滴虫性阴道炎。所有患者RPR均阴性。
表1 PCR检测疑似HSV-Ⅱ DNA结果 （略）
3 讨论
GH临床表现变化多样，可表现为原发性GH、初次发作的非原发性HSV-Ⅱ GH（既往有过HSV-Ⅰ感染）、初次发作的再活动性GH（原有HSV潜伏感染）、复发性GH、免疫缺陷或免疫抑制者（含HIV/AIDS）GH、亚临床型及未识别症状GH、孕妇GH、新生儿HSV感染。典型的临床表现易引起患者及医生的注意，而不典型的临床表现，由于人们对其缺乏认识，易被患者及医生忽视。因此，在临床上应该对这些不典型的疱疹表现加以重视。有资料表明，在STD门诊就诊外阴有裂隙及细小溃疡损害的女性中，有30%可从其生殖标本中分离出HSV-Ⅱ，在HSV-Ⅱ血清抗体阳性的女性中，上述不典型外阴损害多于HSV-Ⅱ血清抗体阴性者，说明HSV-Ⅱ常为这些不典型外阴损害的原因。另外，在HSV-Ⅱ血清抗体阳性者中，60%为未识别症状GH患者；20%为识别症状GH患者，20%为无症状亚临床GH患者，即80%实际上有症状。大多数被认为是亚临床型GH的患者并非真的无症状，只是症状未被发现。因此，所有生殖器溃疡和裂隙均应考虑到GH的可能性，并做相应的实验室检测。
笔者对30例女性生殖器部位具有上述部分不典型皮损的患者利用PCR进行HSV-Ⅱ DNA检测，结果7例HSV-ⅡDNA检测阳性，23例HSV-Ⅱ DNA检测阴性，检测率为23.33%，较国内报道的37.6%较低。其中以裂隙的检测率最高，为33.33%（2/6），其次是硬结，其控制率为25%（1/4），宫颈糜烂的检测率为21.05%（4/15），1例尿道口红肿者未检测到HSV-Ⅱ DNA，HSV-Ⅱ DNA检测阳性者经用阿昔洛韦400mg bid×7天，干扰素100万u肌注每周2次，局部外涂环胞苷膏，治疗后均痊愈，追踪半年未见复发。皮损及症状以裂隙者消退最快，均于用药1天后患者自觉疼痛明显消退，2～3天后裂隙消失。
在本组资料中，笔者感到不足之处是患者未能同时检测HSV-Ⅰ，所以即使是HSV-Ⅱ检测阴性，也不能完全排除并非HSV感染。因为无症状或亚临床症状GH、HSV潜伏感染是GH水平和垂直传播的重要因素，也是GH预防与控制的重要问题。因此笔者认为，在临床工作中，应重视GH不典型的临床表现这个问题，对于生殖器溃疡、裂隙、硬结、非特异性红斑、宫颈糜烂等，均应考虑GH的可能性，争取早期诊断，早期治疗，从而有效地控制GH的传播。
(编辑：唐 城)
作者单位： 434401 湖北石首，石首市东升卫生院
第四节 下丘脑-垂体内分泌功能紊乱的临床生化
一、下丘脑-垂体内分泌功能及调节
㈠垂体分泌的激素
垂体即脑垂体，为位于颅底蝶鞍中的重要内分泌器官，由茎状垂体柄与下丘脑相连。从组织学上可将垂体分做腺垂体及神经垂体。腺垂体包括前部、结节部和中间部，神经垂体由下丘脑某些神经元直接延续而成。垂体分泌的激素相应分做腺垂体激素和神经垂体激素两类。有关激素的生理作用已在生理学中介绍。表12-5概括了重要的垂体激素及其主要生理功能。
上述激素均为肽类或糖蛋白，其中TSH、LH和FSH均是由α和β两个多肽亚基组成的糖蛋白。这三种激素的α亚基具高度同源性，氨基酸残基亦较接近，其生理活性主要取决于β亚基。在用免疫化学法检测时，往往存在交叉免疫反应而互相干扰。
表12-5 主要的垂体激素及生理作用
| 激 素 名 称 | 主要生理作用 |
| 腺垂体激素 | |
| 生长激素（growth hormone，GH） | 促进机体生长 |
| 促肾上腺皮质激素（corticotropin，ACTH） | 促进肾上腺皮质激素合成及释放 |
| 促甲状腺素（thyrotropin，TSH） | 促进甲状腺激素合成及释放 |
| 卵泡刺激素（follicle-stimulating hormone，FSH） | 促进卵泡或精子生成 |
| 黄体生成素（luterzilizing hormone，LH） | 促进排卵和黄体生成，刺激孕激素、雄激素分泌 |
| 催乳素（prolactin，PRL） | 刺激乳房发育及泌乳 |
| 黑色细胞刺激素（melanocyte stimulating hormone，MSH） | 促黑色细胞合成黑色素 |
| 神经垂体激素 | |
| 抗利尿激素（antidiuretic hormone，ADH） | 收缩血管，促进集尿管对水重吸收 |
| 催产素（oxytocin，OT） | 促进子宫收缩，乳腺泌乳 |
㈡下丘脑激素
下丘脑的一些特化的神经细胞可分泌不同的调节腺垂体有关激素释放的调节激素（因子）。从组织结构上看，这些分泌性神经细胞的轴突组成结节-漏斗束，终止于垂体柄内垂体门脉系统的初级毛细血管网周围。借助特殊的垂体门脉系统，这些分泌性神经细胞释放的调节激素，可迅速直接地被输送至腺垂体发挥作用。下丘脑调节激素均是多肽，这些激素的名称、缩写及受其调节的腺垂体激素见表12-6。从表中可看出，下丘脑调节激素的作用通过其名称即可知。但也存在某些交叉，如TRH还可促进生长激素和催乳素释放，而GHIH也能抑制腺垂体TSH、ACTH及胰腺胰岛素的释放。近年还发现，下丘脑外的某些神经细胞及一些脏器组织细胞，也可产生某些下丘脑激素。这些下丘脑外活性多肽的功能尚不清。
表12-6 下丘脑分泌的主要调节激素
| 激素名称 | 调节的腺垂体激素 |
| 促甲状腺激素释放激素（thyrotropin-releasing hormone，TRH） | TSH（主要），GH，PRL，FSH |
| 促性腺激素释放激素（gonandotropin-releasing hormone，GnRH） | LH，FSH |
| 促肾上腺皮质激素释放激素（corticotropin-releasing hormone，CRH） | ACTH |
| 生长激素释放激素（growth hormone-releasing hormone，CRH） | GH |
| 生长激素抑制激素（growth hormone-inhibiting hormone，GHIH） | GH（主要），TSH，ACTH，PRL |
| 催乳素释放激素（prolactin-releasing hormone，PRH） | PRL |
| 催乳素抑制激素（prolactin-inhibiting hormone，PIH） | PRL |
| 黑色细胞刺激素释放激素（melanocyte stimulatinghormone-re-leasing hormone，MRH） | MSH |
| 黑色细胞刺激素抑制激素（melanocyte stimulatinghormone-in-hibiting hormone，MIH） | MSH |
㈢下丘脑-腺垂体激素分泌的调节
下丘脑-腺垂体激素分泌的调节，主要受腺垂体各种促激素作用的靶腺分泌的激素之反馈调节（长反馈），其中甲状腺激素的长反馈调节主要作用于腺垂体，而其他外周激素长反馈调节作用部位则主要为下丘脑水平（图12-1）。前已谈到长反馈调节的主要方式为负反馈，但在月经周期中排卵期前，当雌激素水平达最高峰时，可正反馈地调节下丘脑相关激素的释放（短反馈），在GH分泌的调节中，短反馈为主要方式。而下丘脑激素或腺垂体激素，还可负反馈地调节下丘脑或腺垂体对自身的合成和分泌（超短反馈）。此外，应激状态、某些外周感觉神经冲动以及边缘系统的情绪活动等，均可通过下丘脑以外的中枢神经系统，影响下丘脑-垂体的的激素分泌，并进而影响外周内分泌腺功能。这种神经系统对内分泌的控制，还表现为多种内分泌功能的昼夜节律。
有关下丘脑-腺垂体调节激素的紊乱，将分别在本章有关外周内分泌腺功能紊乱中介绍，而ADH及OT的临床生化分别在第五章和第十四章中讨论。本节主要讨论有关GH紊乱的临床生化，并简单介绍催乳素瘤。
二、生长激素及生长调节素
㈠生长激素的化学、分泌调节及作用
生长激素（growth hormone，GH；somatotropin，STH）是腺垂体嗜酸细胞分泌的，由191个氨基酸残基组成的直链肽类激素。其结构与PRL相似，并有一定交叉抗原性。释放入血液中的GH不与血浆蛋白结合，以游离形式输送到各靶组织发挥作用。Gh 的生理作用最主要的是对成年前长骨生长的促进。现已明确，这一作用是通过生长调节素（so-matomedin，SM）的介导，促进硫酸掺入到骨骺软骨中，及尿嘧啶核苷、胸腺嘧啶核苷分别掺入软骨细胞RNA或DNA中，加速RNA、DNA及蛋白粘多糖合成及软骨细胞分裂增殖，使骨骺板增厚，身材得以长高。GH亦参与代谢调节，主要表现为与生长相适应的蛋白质同化作用，产生正氮平衡；促进体脂水解，血游离脂肪酸升高；对糖代谢则可促进肝糖原分解，升高血糖。此外，GH对维持正常的性发育也有重要作用。
GH的分泌主要受下丘脑GHRH和GHIH的控制。除GH和SM可反馈性调节GHRH和GHIH释放外，剧烈运动、精氨酸等氨基酸、多巴胺、中枢α2肾上腺素受体激动剂等，可通过作用于下丘脑，垂体或下丘脑以外的中枢神经系统，促进GH的分泌。正常情况下，随机体生长发育阶段不同而有不同的GH水平。而每日生长激素的分泌存在昼夜节律性波动，分泌主要在熟睡后1h左右（睡眠脑电图时相3或4期）呈脉冲式进行。
㈡生长调节素
生长调节素（SM）即生长激素依赖性胰岛素样生长调节因子（GH-dependent insulin-like growth factor ，IGF），曾称硫化因子（sulfation factor）。SM为一类在GH作用下，主要在肝脏也由多种GH靶细胞合成的多肽，分子量6000~8500。现至少已确定A、B、C三种亚型，均具胰岛素作用。其中SM-C即IGf I，其结构与胰岛素有近一半的氨基酸残基相同。和其他肽类激素不同，血液中的SM几乎全部均和高亲和力的SM结合蛋白形成可逆结合而运输。如前所述，现至少肯定GH的促生长作用必须通过SM介导，也有认为GH的代谢调节作用也依赖于SM。从这一意义上说，SM水平反映GH的生物活性比GH本身更为直接。
三、生长激素功能紊乱的生化诊断
㈠生长激素功能紊乱
⒈生长激素缺乏症 生长激素缺乏症（growth hormone deficiency）又称垂体性侏儒症（pituitary dwarfism），是由于下丘脑-垂体-GH-SM中任一过程受损而产生的儿童及青少年生长发育障碍。按病因可分做：①原因不明，但可能在胚胎发育或围产期下丘脑损伤，致GHRH合成、分泌不足，或垂体损伤产生的持发性GH缺乏症，约占70％，大多伴有其他垂体激素缺乏症；②遗传性GH缺乏症，以不同的遗传方式所致的单一性GH缺乏为多见，极少数病人也表现为包括GH在内的多种垂体激素缺乏症。近年还发现有少数病人表现为遗传性SM生成障碍，其GH反增多；③继发性GH缺乏症，由于下丘脑、垂体及周围组织的后天性病变或损伤，如肿瘤压迫、感染、外伤、手术切除等，致GH分泌不足。
GH缺乏症的突出临床表现为生长发育迟缓，身材矮小，但大多匀称，骨龄至少落后2年以上。若未伴甲状腺功能减退，则智力一般正常，以别于呆小症。此外性发育迟缓，特别是伴有促性腺激素缺乏者尤显。患儿大多血糖偏低，若伴ACTH缺乏者更显著，婴幼儿甚至可出现低血糖抽搐、昏迷。
⒉巨人症及肢端肥大症 巨人症（gigantism）和肢端肥大症（acromegaly）由GH过度分泌而致。若起病于生长发育期表现为前者，而在成人起病则表现为后者，巨人症多可继续发展为肢端肥大症。病因大多为垂体腺瘤、癌或GH分泌细胞增生而致；也有少数系可分泌GHRH或GH的垂体外肿瘤产生的异源性GHRH或GH综合征，包括胰腺瘤、胰岛细胞癌、肠及支气管类癌等。单纯巨人症以身材异常高大、肌肉发达、性早熟为突出表现。同时存在高基础代谢率、血糖升高、糖耐量降低、尿糖等实验室检查改变。但生长至最高峰后，各器官功能逐渐出现衰老样减退。肢端肥大症者由于生长发育已停止，GH的促骨细胞增殖作用表现为骨周增长，产生肢端肥大和特殊的面部表现，及包括外周内分泌腺在内的广泛性内脏肥大。亦有高血糖、尿糖、糖耐量降低、高脂血症、高血清钙等实验室检查改变。粥样动脉硬化及心衰常为本病死因。病情发展至高峰后，亦转入同巨人症一样的衰退期。
㈡GH紊乱的临床生化检查
⒈血清（浆）GH测定均用免疫化学法测定。一般在清晨起床前，空腹平卧安静状态下取血测定作为基础值。正常参考范围为新生儿15-40μg/L，2岁儿童平均约为4μg/L，2-4岁儿童平均约8μg/L，4岁以上儿童及成人为0-5μg/L，女性略高于男性。若测定结果远远超出正常水平，结合临床所见，有助于巨人症或肢端肥大症、以及遗传性SN生成缺陷所致的GH缺乏症诊断。但由于前述GH每日分泌主要在夜间熟睡中，呈脉冲式释放的生理性波动特点，而其半寿期又仅20min。因此若在非脉冲式释放期取样测定，GH水平再低也无多大意义。故在诊断GH缺乏症时，最好在病儿熟睡后1-1.5h取血测定。更为严格的是插入留置式取血导管后，进行24h或晚8点到次晨8点内每0.5h取血测定Gh ，了解全天或夜间GH分泌的总体情况。若测定结果为低，则还需应用下列兴奋试验证实。
⒉动态功能试验 GH释放的兴奋试验，方法较多，常用的有以下几种：
⑴运动刺激试验：可合作年龄儿童空腹取血作基础对照后，剧烈运动20-30min，运动结束后20-30min取血测定。因剧烈运动及可能存在的血糖水平偏低均可刺激腺垂体释放GH，故运动后，正常者血清GH值应较基础对照值明显升高或≥10μg/L；GH缺乏症者，运动后GH水平＜5μg/L。
⑵药物刺激试验：可刺激腺垂体释放GH的药物很多，目前常用的药物及方法为：①胰岛素-低血糖试验（insulin-hypoglycemia test），因低血糖应激状态可刺激腺垂体释放GH、ACTH、PRL等多种激素，故在清晨空腹卧床采血作对照后，按0.1μ/kg体重静脉注射普通胰岛素后30、60、90、120及150min分别取血，测定GH水平，必要时可同时检测ACTH、PRL，以发现复合性垂体前叶功能减退；②其他药物刺激试验均是在同上抽取清晨卧床空腹血后，给予L-多巴（促GHRH释放）500mg（儿童10mg/kg体重）一剂口服，或可乐定（促GHRH释放）4μg/kg体重（或150μg/m2体表面积）一剂口服，或盐酸精氨酸（促进垂体释放GH）0.5g/kg在30min内静脉滴注。分别在上述药物使用后30、60、90和120min取血，测定血清（浆）GH水平。正常人在使用上述刺激剂后，GH分泌峰多在60或90min出现，胰岛素可推迟到120或150min出现，峰值应比对照基础值升高5-7μg/L以上，或峰值浓度≥20μg/L。若两项以上刺激试验峰浓度均＜5μg/L，则为GH缺乏症。而峰值浓度≥20μg/L，则可排除GH缺乏症，但GH受体缺陷等所致SM遗传性生成障碍者，GH基础值反可升高，并且对上述兴奋试验可有正常人样反应，此时只有通过SM测定进行鉴别。
⒊GH分泌的抑制试验对于多次测定基础GH值约＞10μg/L的疑为巨人症或肢端肥大症者，应考虑进一步作高血糖抑制GH释放试验。即按上述方法抽取空腹基础静脉血，口服含100g（儿童1.75g.kg体重）葡萄糖的浓糖水后，分别在30、60、90和120min取血，测定各血清GH水平。正常人服用葡萄糖后血清GH最低应降至2μg/L以下，或在基础对照水平50％以下。垂体腺瘤性或异源性GH所致巨人症或肢端肥大症者，因呈“自主性”GH分泌，不会被明显抑制，最低浓度＞5μg/L，或在基础对照水平50％以上。但本试验可有假阴性出现，特别在治疗可能出现的高血压、高血糖，使用了可乐定、α-甲基多巴等中枢α2肾上腺素受体激动剂或降血糖药者，应注意避免，最好停用上述药物一周以上再行本试验。
⒋SM-C及SM结合蛋白测定前面已介绍GH的促生长作用等，均需通过SM介导。SM为一类多肽物质，现已分离出A、B、C三亚型。其中SM-C（IGf I）为中性肽，血浆中SM-C几乎均为GH作用于肝细胞膜受体后，诱导肝细胞合成释放的。由于SM-C的血浆浓度不随GH分泌的脉冲式波动而变动，前述各种刺激或抑制GH释放的因素，均不能在短时内引起SM-C的浓度改变，因此其水平较稳定。单次取样测定即可了解较长一段时间的GH功能状况。再加之其半寿期长，血浆浓度高，易于检测。现在推荐以免疫化学法检测单次血样中SM-C浓度，作为判断GH功能状况的简便而可靠的筛选方法。血清SM-C正常值参考范围为：青春期前儿童0.1-2.8u/ml，青春期少年0.9-5.9u/ml，成人男性0.3-1.9u/ml，女性0.5-2.2u/ml。任何GH缺乏症，包括高GH水平的遗传性GH受体缺陷性者，SM-C均低于同龄正常水平的下限；巨人症及肢端肥大症者则远远高于正常水平。但恶病质、严重营养不良及严重肝病者，SM-C可降低；青春期少年有时可超出正常值上限。
由于血浆中SM几乎均与高亲和力的特异结合蛋白SMBP（IGFBP）结合，亦有主张检测SMBP间接反映GH功能者，但尚未成熟。
四、催乳素瘤
催乳素瘤（prolactinoma，PRL瘤）为功能性垂体腺瘤中最常见者，好发于女性，多为微小腺瘤，临床表现为泌乳、闭经、多毛、痤疮及不育等。男性则往往为大腺瘤，临床以性功能减退、阳萎、不育及垂体压迫症状为主，偶见泌乳。临床生化检查可见血清PRL极度升高。正常人清晨血清基础值男性＜20μg/L，非妊娠及哺乳期女性即便在月经周期的黄体期亦＜40μg/L。不论男女，若血清PRL基础值＞200μg/L，应高度怀疑本病，＞300μg/L则可确诊。对于PRL水平在100-300μg/L的高催乳素状态者，可以TRH、氯丙嗪或灭吐灵兴奋试验协助鉴别。正常人及功能性高催乳素血症者，上述兴奋试验可使血清PRL较基础对照明显升高，而催乳素瘤者因呈“自主性”高分泌，故反应低下或无，即便有弱反应也峰值推迟。
