March 7, 2012 -- New research holds the promise of freeing many organ transplant patients from a lifetime of anti-rejection drugs.
In the first study of its kind, eight kidney transplant patients received stem cells from their kidney donors manipulated to “trick” their bodies into accepting the foreign organ as its own.
Transplant recipients who are not perfectly matched with their donors typically take several drugs a day for the rest of their lives to keep their bodies from rejecting the new organ and to treat the side effects of those drugs.
Lindsay Porter, who was the last of the eight patients enrolled in the new study, had her kidney transplant in the summer of 2010 and was weaned off all anti-rejection drugs within a year.
The Chicago actress and mother says she feels better than she has in 15 years and sometimes has to remind herself that she had a kidney transplant.
“I was 45 when I had the surgery, and I knew I would probably need another kidney at some point,” she tells WebMD. “The opportunity to have a transplant that would last for the rest of my life and to avoid all of those drugs was very appealing.”
The ongoing research is the culmination of many years of work by researcher Suzanne Ildstad, MD, of the University of Louisville, and other researchers, including transplant surgeon Joseph Leventhal, MD, PhD, of Chicago’s Northwestern University.
The new wrinkle is that organ donors who are not a perfect genetic match with the patient donate blood as well as a kidney for the procedure.
Bone marrow stem cells collected from the blood were processed in an 18-hour procedure to remove cells associated with organ rejection, leaving behind “facilitating” cells that do not promote rejection, Ildstad says.
Porter and the other patients in the study had chemotherapy about a month before their surgeries to suppress their own immune systems before receiving the manipulated donor stem cells in an effort to increase the likelihood that those stem cells would reprogram the body to accept the transplant.
The procedures were performed in the eight patients between February 2009 and July 2010, and five of the eight have maintained normal kidney function and were able to stop taking all anti-rejection drugs within a year of having their transplants.
Ildstad says the stem cell approach may prove useful for other solid-organ transplants and for many other conditions, including type 1 diabetes and sickle cell anemia.
Leventhal says the research team is now working to modify the approach so that it can be used when the transplanted kidney comes from a donor who has died.
About two-thirds of the roughly 17,000 kidney transplants performed in the U.S. each year involve deceased donors.
【摘要】 目的 探讨过敏性紫癜（HSP）病儿急性期免疫细胞功能变化及其意义。方法 以53例HSP急性期病儿为研究对象，并根据有无尿检异常分为肾炎（HSPN）组和非肾炎（NHSPN）组，以30例正常儿童作为对照组。采用流式细胞术检测3组外周血CD3+细胞、CD4+细胞、CD8+细胞、CD4+/CD8+比值、CD4+CD25+细胞、NK细胞（CD16+CD56+）和B淋巴细胞（CD19+）水平。结果 HSPN组和NHSPN组外周血CD3+细胞、CD4+细胞、CD4+/CD8+比值、CD4+CD25+ 细胞和NK细胞水平较对照组显著降低，CD8+细胞和B淋巴细胞水平较对照组显著增高(F=6.80～46.40，q=2.81～4.25,P<0.01、0.05)，而HSPN组和NHSPN组间比较差异无统计学意义(P>0.05)。HSP病儿CD4+CD25+细胞水平与CD4+细胞、CD4+/CD8+比值呈正相关(r=0.369、0.285,P<0.01、0.05)，与CD19+细胞呈负相关(r=-0.279,P<0.05)，而与CD8+细胞和CD16+CD56+细胞无相关性(r=0.009、-0.104,P>0.05)。结论 HSP病儿急性期存在免疫细胞功能异常，T细胞亚群紊乱，CD4+CD25+T细胞和NK细胞数量降低，导致B细胞呈多克隆活化，在HSP发病机制中具有重要作用。
【关键词】 紫癜,过敏性 儿童 免疫,细胞 流式细胞术
FUNCTIONAL CHANGES OF IMMUNE CELLS IN CHILDREN WITH HENOCHSCHONLEIN PURPURA IN ACUTE STAGEBAI CUI,
ZONG JINBAO, ZHANG QIUYE
(Department of Pediatrics, The Affiliated Hospital of Qingdao University Medical College, Qingdao 266003, China);
［ABSTRACT］ Objective To investigate the functional changes of immune cells in children with HenochSchonlein Purpura（HSP） in acute stage and their significance. Methods Fiftythree children with HSP were involved in this study, who were divided into HenochSchonleinPurpuranephritis (HSPN) group and nonHSPN (NHSPN) based on whether the urine analysis was normal or not. Thirty healthy children were served as control. Flow cytometry was applied to detect the amounts of CD3+ cells, CD4+ cells and CD8+ cells, CD4+ /CD8+ ratio, and the levels of CD4+CD25+ cells, NK cells (CD16+CD56+) and B cells (CD19+) in peripheral blood. Results The amounts of CD3+ cells, CD4+ cells, CD4+CD25+ cells, NK cells and the ratio of CD4+/CD8+ in the HSP and NHSPN children were significantly lower than those in the control children, while CD8+ cells and B cells were significantly higher (F=6.80-46.40;q=2.81-4.25;P<0.01,0.05). The differences between those in HSP and theNHSPN children were not significant (P>0.05). The amount of CD4+CD25+ cells of the HSP was positively correlated with that of CD4+ cells and CD4+/CD8+ ratio (r=0.369,P<0.01;r=0.285,P<0.05), and negatively correlated with tthat of CD19+ cells (r=-0.279,P<0.05), but not correlated with those of CD8+ cells and CD16+CD56+ cells (r=0.009,-0.104;P>0.05).Conclusion Children with HSP in acute stage have a disordered cellmediated immunity, which mainly manifests as derangement of Tcell subsets, decrease of CD4+CD25+ T cells and NK cells, resulting in polyclonal Bcell activities, which plays an important role in the pathogenesis of this condition.
［KEY WORDS］ purpura, SchoenleinHenoch; child; immunity, cell; flow cytometry
过敏性紫癜(HSP)是儿童时期常见的血管炎性疾病，以广泛的小血管炎症为病理基础，肾脏损害是其常见表现和并发症，其病因和发病机制尚未完全明确。近年来的研究结果表明，HSP病儿存在免疫调节功能紊乱[1]。本文采用流式细胞术(FCM)检测HSP急性期病儿外周血T淋巴细胞及亚群（包括CD3+细胞、CD4+细胞、CD8+细胞、CD4+CD25+细胞)、B淋巴细胞(CD19+)和NK细胞（自然杀伤细胞，CD16+CD56+）水平变化，进一步探讨HSP发病机制。现将结果报告如下。
1 资料与方法
1.1 一般资料
1.1.1 HSP组
选取2008年1月—2009年4月在我院儿内科住院的HSP急性期病儿53例，男31例，女22例，年龄2～12岁，平均8.65岁，均符合《诸福棠实用儿科学》HSP的诊断标准。依据有无尿检异常[血尿和（或）蛋白尿]将HSP组病儿分为肾炎（HSPN）组和非肾炎（NHSPN）组。肾炎组18例，男10例，女8例，年龄3～12岁，平均7.33岁；非肾炎组35例，男21例，女14例，年龄2～12岁，平均9.97岁。采血前均未接受糖皮质激素或免疫调节剂治疗。
1.1.2 对照组
选取同期我院儿童保健门诊健康体检儿童30例，男16例，女14例，年龄1～12岁，平均8.30岁，均无近期感染及个人或家庭过敏史、系统性疾病史。
1.2 检测方法
采用直接免疫荧光标记全血溶血方法，在流式细胞仪(美国Coulter公司)上测定外周血CD3+、CD4+、CD8+、CD4+CD25+、CD19+、CD16+CD56+细胞数，并计算CD4+/CD8+比值。取肝素抗凝外周血100 μL, 分别加CD3FITC/CD16+56PEz、CD4FITC/CD8PE、CD3FITC/CD19PE、CD4FITC/CD25PE及FITC/PE Mouse IgG1双标记荧光抗体20 μL(抗体均购于法国Immunotech公司),混匀，室温避光孵育20 min，然后加入红细胞裂解液1 mL，混匀，室温避光孵育20 min，用PBS洗涤2次后上机检测。
1.3 统计学处理
应用SPSS 17.0及PPMS 1.5[2]统计软件进行统计处理。实验数据呈正态分布，以±s表示。各组间比较采用单因素方差分析及q检验，两变量间相关分析采用直线相关分析法。
2 结果
2.1 HSP病儿急性期外周血淋巴细胞及其亚群水平变化
HSPN组和NHSPN组病儿外周血CD3+细胞、CD4+细胞、CD4+/CD8+细胞比值、CD4+CD25+ 细胞、CD16+CD56+细胞水平较对照组显著降低，CD8+细胞、CD19+细胞水平较对照组显著增高(F=6.80～46.40，q=2.81～4.25,P<0.01、0.05)，而HSPN组和NHSPN组间淋巴细胞及其亚群比较差异无统计学意义(P>0.05)。见表1。表1 各组外周血淋巴细胞及其亚群检测结果比较(略)
2.2 HSP病儿CD4+CD25+细胞计数与其他淋巴细胞水平变化的相关性
HSP病儿CD4+CD25+细胞水平与CD4+细胞、CD4+/CD8+比值呈正相关(r=0.369、0.285,P<0.01、0.05)，与CD19+细胞水平呈负相关(r=-0.279,P<0.05)，而与CD8+细胞和CD16+CD56+细胞则无相关性(r=0.009、-0.104,P>0.05)。
3 讨论
HSP是一种主要累及毛细血管的变态反应性疾病，以非血小板减少性紫癜、关节炎或关节痛、腹痛、胃肠道出血及肾炎为主要临床表现，其中肾损害的严重程度直接影响到该病的预后。其发病机制尚不完全清楚。多数研究表明，HSP与免疫调节功能紊乱有关。在免疫应答过程中，CD4与CD8淋巴细胞起着十分重要的调节作用。在正常情况下，CD4和CD8相互诱导，相互制约形成T细胞网络，从而调节正常免疫功能和维持免疫的自稳性。本文结果显示，HSPN组和NHSPN组急性期病儿CD3+细胞、CD4+细胞、CD4+/CD8+比值显著降低，CD8+细胞明显升高，与WIERCINSKI等[3]报道结果相符，表明HSP病儿存在细胞免疫功能失调，主要表现为辅助T淋巴细胞(CD4+)数量降低，效应T淋巴细胞(CD8+)数量增高，CD4+/CD8+比值下降。但有研究指出，HSP病儿急性期T细胞亚群变化与HSP是否并发早发性肾损害无明显相关性[4]。本文结果亦显示，HSP病儿急性期B淋巴细胞（CD19+）显著升高，而NK细胞（CD16+CD56+）显著下降。CD19是成熟B淋巴细胞特异性标志，CD19显著升高，说明B淋巴细胞活化增殖增多，体液免疫反应增强。有研究指出，病毒或细菌感染和动物性食物蛋白过敏是HSP最主要诱因[5]，在抗原物质刺激下可使B细胞数量增加。B细胞增殖与Th2细胞功能亢进有关，研究发现HSP病儿Th2类细胞因子如IL4和IL6显著增高，Thl类细胞因子如IL2水平降低[67]。结合本实验结果，考虑B细胞增高与T细胞亚群紊乱及其分泌的细胞因子异常有关。CD16+CD56+是鉴别NK细胞的特征性标志。NK细胞是一类重要的免疫调节细胞，对T细胞、B细胞等均有调节作用。本研究结果与有关文献报道一致[8]。NK细胞的降低对B淋巴细胞增长分化的抑制力减弱，导致B淋巴细胞数量及活性升高，分泌免疫球蛋白增加，参与免疫复合物形成而引起毛细血管壁的损伤[9]。本文结果显示，HSPN组与NHSPN组相比较外周血CD3+细胞、CD4+细胞、CD8+细胞、CD4+/CD8+比值、CD4+CD25+细胞、NK细胞和B淋巴细胞水平差异均无显著性，提示HSP急性期有无肾脏损害病儿间外周血淋巴细胞数量变化并无差异。齐鲁医学杂志2010年4月第25卷第２期 Med J Qilu, April 2010, Vol.25, No.２
调节性T淋巴细胞（Tregs）是T淋巴细胞的重要亚群，依据其产生部位和生成方式分为自然产生的Tregs和适应性Tregs，CD4+CD25+T细胞即自然产生的Tregs，是机体内Tregs的主要类型,占人外周血CD4+T细胞总数的5%～10%。CD4+CD25+ Tregs具有免疫无能和免疫抑制两大功能特征，主要通过细胞接触机制抑制CD4+细胞、CD8+细胞的免疫反应[10]。越来越多的证据表明，CD4+CD25+ Tregs在抑制自身反应T细胞的活化和增殖方面起着不可或缺的作用。去除CD4+CD25+ Tregs的动物会出现各种自身免疫病如甲状腺炎、胃炎、结肠炎和1型糖尿病，而重新被动输注正常的CD4+CD25+ Tregs可预防这些疾病的发生[11]。此外，CD4+CD25+ Tregs对肿瘤免疫、微生物免疫、抑制变态反应以及骨髓移植排异反应均有重要的调控作用。本研究结果显示，HSP病儿急性期CD4+CD25+细胞显著下降，与有关文献报道一致[1213]。HSP病儿CD4+CD25+细胞与CD4+细胞、CD4+/CD8+比值呈正相关，而与CD19+细胞呈负相关，提示CD4+CD25+ Tregs异常在HSP的发病机制中具有重要的意义。其机制可能涉及以下几个方面。①CD4+CD25+ Tregs表面表达的CD25、细胞毒性T淋巴细胞相关抗原(CTLA4)、膜性TGFβ等与效应T细胞接触后,抑制T细胞生长因子IL2的基因表达,抑制抗原特异性CD4+及CD8+T细胞增生,或使其转变为无能或反应低下的T细胞,使机体产生免疫耐受[1415]。当该类细胞减少或功能降低时,免疫耐受平衡破坏,机体接受抗原刺激后,发生变态反应,产生免疫复合物沉积, 则可能引起HSP。②CD4+CD25+ Tregs使专职抗原提呈细胞(APC)表面的协同刺激分子减少,抗原提呈功能减弱,不能有效激发特异或非特异性免疫应答[16]。当CD4+CD25+ Tregs减少或功能降低时,APC抗原提呈功能增强,使机体对变应原发生超敏反应。③CD4+CD25+ Tregs可产生较多抑制性细胞因子IL10, IL10在体内外能调节抗原特异性B细胞,调节抗体类别转换,抑制变态反应。当该细胞数量或功能降低时, IL10减少,则可能发生变态反应性疾病。
综上所述，HSP病儿急性期存在免疫细胞功能异常，主要表现为T细胞亚群紊乱（CD4+/CD8+比值降低）、CD4+CD25+T细胞和NK细胞数量降低，导致B细胞呈多克隆活化，在HSP发病机制中具有重要作用，提示免疫调节疗法在HSP治疗中有重要价值。
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March 4, 2011 -- Using stem cells, researchers have re-created key neurons affected by Alzheimer’s disease in the lab, a discovery that could lead to improved treatments for the disease.
Researchers say it’s the first time embryonic stem cells have been transformed into a critical type of cell that dies off in the early stages of Alzheimer’s disease and is implicated in memory loss.
The cells, known as basal forebrain cholinergic neurons, help the area of the brain known as the hippocampus retrieve memories in the brain, an ability that is lost in early Alzheimer’s disease.
Researchers say there are a limited number of these cells in the brain and their loss in Alzheimer’s disease has a rapid and devastating effect on memory.
“Now that we have learned how to make these cells, we can study them in a tissue culture dish and figure out what we can do to prevent them from dying,” says researcher Jack Kessler, MD, chair of neurology at Northwestern University Feinberg School of Medicine, in a news release.
Researchers say the ability to grow these cells in the lab will allow them to quickly test different drugs to see which ones keep the cells alive. In addition, the stem-cell-derived neurons may eventually be transplanted directly into the brains of people with Alzheimer’s disease to potentially reverse memory loss.
In the study, published in the journal Stem Cells, researchers describe a new method of duplicating the neurons using human embryonic stem cells.
Stem cells have the ability to develop into any type of cell in the body, and researchers had to grow and test millions of cells to determine how to activate the exact sequence of genes to transform the cells into the desired cell.
They then transplanted the new cells into the brains of mice to see if they functioned normally. The results showed the neurons produced connecting fibers or axons to the hippocampus and produced acetylcholine, a chemical required by the hippocampus in order to retrieve memories.
Dec. 10, 2010 (San Antonio) -- Women with metastatic breast cancer who have no tumor cells circulating in their blood after the first round of treatment live longer than those who do, French researchers report.
Circulating tumor cells, or CTCs, are cells that break off from a tumor and escape into the bloodstream. These cells can travel to other organs and establish new tumors.
Several studies have shown that higher levels of CTCs are associated with an increased risk for recurrence and death in metastatic breast cancer patients.
But the new study is the largest to look at the topic, and the CTCs predicted prognosis even after taking other markers of survival into account, says Jean-Yves Pierga, MD, PhD, professor of the medical oncology department at Institut Curie and Universite Paris Descartes, France.
The study also suggests CTCs can be used to monitor whether a woman is responding to treatment, he tells WebMD.
Pierga reported the findings at the annual San Antonio Breast Cancer Symposium.
The study involved 267 women whose cancer had spread to other parts of their body (metastasized) and who were receiving chemotherapy with or without the targeted drug Avastin for the first time.
Before treatment, two-thirds of the women had one or more CTCs; 44% had five or more CTCs.
After two years, cancer had progressed in about 95% of those with five or more CTCs, compared with about 70% of those with fewer or no CTCs.
Looked at another way, women with five or more CTCs were 90% more likely to have their cancer worsen and nearly two-and-a-half times more likely to die, even after other tumor markers were taken into account, Pierga says.
Then the researchers looked at women's CTC levels after their first round of treatment. At two years, cancer had progressed in about 70% of those with fewer than five CTCs, but in all of those with more CTCs. Over 90% of those with fewer than five CTCs were still alive, compared with less than half of the women with more CTCs.
The study also showed that after three rounds of treatment, CTCs dropped more in women who received Avastin plus chemotherapy, compared with those who just got chemo.
Still, doctors appear divided on whether CTC testing is useful.
Alison T. Stopeck, MD, director of the Clinical Breast Cancer Program at the Arizona Cancer Center in Tucson, says she doesn't use the CTC test at all.
"More often than not, I know which of my patients with metastatic breast cancer are progressing just by examining them," she tells WebMD.
Plus a high CTC count doesn't provide any information about what treatment might help, Stopeck says.
Minetta Liu, MD, director of translational breast cancer research at Georgetown Lombardi Comprehensive Cancer Center in Washington D.C., says the test is very useful.
"I use every tool I have," she tells WebMD. In some cases, knowing a woman has no CTCs allows her to delay much more expensive imaging tests to track the progress of the cancer, Liu says.
Nov. 16, 2010 (Chicago) -- Stem cells taken from belly fat may be able to boost cardiac function after a heart attack, preliminary research suggests.
In a study of 14 people who had a heart attack, fat-derived stem cells reduced the amount of damaged heart tissue, increased blood flow in the heart, and improved the heart's pumping ability, compared with placebo.
Due to the study's small size, however, the difference between the two groups could have been due to chance.
"But given the dramatic and consistent results, we think it is a real effect," says study head Eric Duckers, MD, PhD, of Erasmus University Medical Center in Rotterdam, Netherlands.
U.S. stem cell researcher Douglas Losordo, MD, of the program in cardiovascular regenerative medicine at Northwestern Memorial Hospital in Chicago, tells WebMD, "The evidence strongly suggests fat stem cells can stimulate the repair process after a heart attack. But these are still early days. We have to await the results of larger randomized trials [pitting placebo against fat stem cells] to determine if the method improves quality of life and extends lives."
It's not the first time heart attack patients have been treated with stem cells. But previous studies used bone marrow stem cells, Duckers says. "The advantage of fat-derived cells is the ease with which you can get them."
"You don't get enough stem cells from bone marrow, so you have to culture them in the lab, a process that can take six to eight weeks," Duckers tells WebMD. He estimates that the 40 cubic centimeters of bone marrow typically removed yields about 25,000 stem cells.
In contrast, just 100 cubic centimeters of fat tissue -- about a half a coffee cup -- contains 2 million stem cells, he says. "With that many cells, you can isolate them and go straightway back to the patient."
In the study, the first of its kind, people were treated within 24 hours of their heart attack after undergoing cardiac catheterization to assess blood flow and angioplasty to open up the blocked heart artery and restore blood flow.
The findings were presented here at the American Heart Association's Scientific Sessions 2010.
The researchers liposuctioned fat from the abdomen of 10 patients, isolated 20 million stem cells, and infused them back into the patient through a catheter -- all in 10 minutes. The other four patients got placebo infusions.
By six months later, SPECT imaging showed that blood flow to the heart had improved 3.5-fold in people getting fat stem cells, compared with those getting placebo. Heart pumping ability increased 5.7% in the stem cell group.
As seen on MRI scans, the average area of heart muscle scarring was cut in half in the treatment group, from 31.6% after the heart attack to 15.4%.
Aug. 30, 2010 (Stockholm, Sweden) -- Giving people with chronic heart failure injections of their own bone-marrow stem cells appears to improve their heart function and extend their lives, new research suggests.
The benefits of the stem cell treatment were apparent within three months and persisted for the five years the patients were followed, says researcher Bodo-Eckehard Strauer, MD, of Heinrich Heine University in Dusseldorf, Germany.
This isn't the first time doctors have reported that stem cells may help improve the health of people with heart failure or other heart conditions.
But the 391-patient study is one of the biggest tests to date of stem cell therapy for heart disease -- and the first to show that the treatment cuts the risk of death in chronic heart failure, Strauer tells WebMD.
The treatment "has almost no risks and is effective when used on top of other treatments for chronic heart failure," he says.
The findings were reported here at the European Society of Cardiology Congress.
One major cause of heart failure occurs when the heart muscle becomes scarred and loses its ability to pump enough blood throughout the body, often after a massive heart attack.
"The hope is that by injecting stem cells into the scarred area, you will bring life back to that area and induce healthy muscle," says American Heart Association spokeswoman Mariell Jessup, MD, medical director of the Penn Heart and Vascular Center at the University of Pennsylvania.
Stem cells are at an early stage of maturation and therefore have the potential to become many different types of cells, including those in the heart muscle.
In the study, bone marrow stem cells were taken from the area at the top of the patient’s pelvic bone. Then they were processed in the lab in such a way as to allow them to be injected into the scarred heart muscle.
Nearly five years after the study started, seven of the 191 patients who had the stem cell treatment had died vs. 32 of 200 patients who did not have the treatment -- a substantial difference.
The stem cell treatment improved the heart's ability to pump blood and restored blood flow to oxygen-starved heart muscle. Patients were able to exercise more. They also reported improved quality of life, Strauer says.
No patient experienced side effects, he says. All patients continued to receive optimal medical treatment throughout the study.
"There's been ongoing excitement about using stem cells to treat heart disease for some time and this study certainly adds to it," Jessup tells WebMD.
But the therapy is not ready for prime time, she says. One of the reasons: In the study, people knew whether they were getting the stem cell treatment, she says.
"It's not like the traditional randomized, controlled trial where people don't know whether they are getting the experimental treatment. That's what we really need," Jessup says.
Also, there may be "some increase in potentially life-threatening [irregular heartbeats]. You can't discount that and say there are no risks," she says.
This study was presented at a medical conference. The findings should be considered preliminary as they have not yet undergone the "peer review" process, in which outside experts scrutinize the data prior to publication in a medical journal.
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【摘要】 目的 探讨体外定向诱导胚胎干细胞(ESCs)分化为甲状腺细胞的可行性。 方法 E14小鼠ESCs诱导发育为拟胚体，再逐步添加促甲状腺素（TSH）、胰岛素（insulin）、KI等共培养。观察细胞分化过程的形态学变化，并以体外培养的成年小鼠甲状腺细胞为阳性对照；RT-PCR法检测甲状腺细胞分化相关基因TSHR、PAX8、NIS、TPO、Tg等表达情况；激光共聚焦显微镜双色荧光检测甲状腺细胞分化标记物PAX8和TTF-1的共表达;在荧光检测得甲状腺细胞分化率的基础上，送检电镜观察分化细胞的超微结构。 结果 诱导培养第6d分化细胞中有甲状腺细胞特有基因PAX8、NIS、TPO、Tg、TSHR的表达；第8d检测到分化细胞中甲状腺细胞标记物TSHR、TTF-1、 PAX8、TTF-2 的表达；细胞形态类似对照甲状腺细胞。 结论 ESCs经胚胎体发育阶段，在特定条件下可定向分化为甲状腺细胞。
【关键词】 干细胞；分化；甲状腺；拟胚体
Experimental study on directed differentiation of embryonic stem cells into thyrocyte-like cells in vitro.
ZHANG Hong, JIANG Ning-yi, LIU Sheng, et al.
( Nuclear Medicine Department, the Second Affiliated Hospital of Sun Yat-sen University,Guangzhou 510120, Guangdong, P. R.China )
Abstract: Objective To investigate the feasibility of directed differentiation of embryonic stem cells (ESCs) into thyrocyte-like cells in vitro. Methods Murine E14 ESCs were cultured in methylcellulose semisolid medium to form embryoid bodies (EBs). These EBs were transferred for further inductive culture with the stepwise addition of growth factors-TSH, insulin and KI into the culture medium. During differentiation, cell morphology was observed through phase contrast microscopy and compared with the normal thyroid cells from mouse. The molecular markers of thyroid cells were performed by indirect immunofluorescsent analysis under fluorescsent microscopy. Gene expressions of thyroid specific mRNA were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) for molecules TSHR, PAX8, NIS, TPO and Tg. Results After EBs formation, on day 6 of further culture added with inductive factors TSH,insulin and KI, ESCs-derived cells expressed thyroid-specific genes such as PAX8, NIS, TPO, Tg and TSHR. On the eighth day, these ESCs-derived thyrocyte-like cells expressed TSHR, TTF-1, PAX8 and TTF-2. Morphology of these markers positive differentiated cells was similar to those normal thyroid cells. Conclusion ESCs can differentiate into thyrocyte-like cells under given inductive conditions and related growth factors in vitro.
Key words: Stem cells; Differentiation; Thyroid gland; Embryoid body
近年来，甲状腺疾病的发生率逐年升高，各种治疗方法如甲状腺肿瘤的手术，有些涉及到甲状腺全切除或次全切除，导致术后甲状腺功能减退；甲状腺机能亢进症的放射性核素131I治疗疗效确切，但其最大的不足是出现甲状腺功能减低症；另外，先天性因素导致的甲状腺疾病日渐增多，如先天性甲状腺功能减低症，发病率大约在1/3000-1/4000[1]之间。如何避免这些甲状腺功能低下患者永久性服用甲状腺素等，并提高其生活质量，是当前亟待解决的问题。目前关于胚胎干细胞(embryonic stem cells, ES)的研究不断增多，技术也不断完善，这为治疗甲状腺功能低下提供了新的研究思路。
本研究旨在探讨ESCs体外定向诱导分化为甲状腺细胞的可行性和分化相关的调节因子，为ESCs源性甲状腺细胞替代治疗甲状腺功能低下提供基础资料。
1 材料与方法
1．1 材料
1．1．1 E14小鼠ESCs细胞株由美国哈佛大学Dr.Xu教授惠赠，中山大学附属第二医院脐血库保存。Balb/c孕鼠及昆明种小鼠购自中山大学实验动物中心。
1．1．2 细胞培养试剂 高糖DMEM培养基、IMDM培养基、RPMI-1640培养基、甲基纤维素、非必需氨基酸、L-谷氨酰胺，（Gbico）ESCs培养专用胎牛血清、0.25%胰蛋白酶含0.04% EDTA)，(Hyclone硫代甘油、明胶、促甲状腺素(TSH)、 维生素C(VitC)、 胰岛素(insulin)、KI、促甲状腺素受体(TSHR)抗体、anti-rabbit IgG cy3 conjugate，（Sigma）重组小鼠白血病抑制因子(rmLIF)（Chemicon），Trizol、Taq酶、dNTP、ThermoScriptTMRT-PCR System(Invitrogen), PAX8抗体、TTF-1抗体、TTF-2抗体。
1．2 方法
1．2．1 鼠胚成纤维细胞（MEF）饲养层的制备 取孕期12.5～14.5d的Balb/c孕鼠，拉颈处死，无菌条件下剥出鼠胚，除去头、尾、四肢和内脏，洗去血污，剪成1～3mm3组织块，移至一无菌离心管内， PBS洗至液体基本无色，吸去PBS，加入2～3倍体积0.25% 胰蛋白酶-0.04% EDTA消化液进行消化， 37℃振荡10～15min，中止消化，静置使组织块沉降，吸取上部细胞悬液至无菌试管中，重复消化剩余胚胎组织。将收集的细胞悬液离心（1000r/min,5min），弃上清夜，加入含10%特级胎牛血清的H-DMEM培养液，重悬细胞，分种至25cm2 的细胞培养瓶中（ρ=5×106个/ml），置37℃、5%CO2培养箱中静置培养。待细胞铺满培养瓶时，进行消化传代。获得的细胞在5代内用作饲养层。
1．2．2 E14细胞的培养传代及脱饲养层培养 E14胚胎干细胞可直接接种于上述MEF细胞上培养传代，培养基为含15% ESCs专用血清的高糖DMEM培养基，添加1.5×10-4mol/L硫代甘油、0.1mmol/L非必需氨基酸、2mmol/L L-谷氨酰胺及双抗。脱饲养层培养时，将细胞接种于铺有0.1%明胶的培养瓶中，培养液同上，但添加2000u/ml的rmLIF。
1．2．3 拟胚体（Embryoid Bodies，EBs）形成及定向诱导 将E14小鼠ESCs以2×104个/ml的密度接种于60mm细菌培养皿悬浮培养6d，培养基换为含15% ESCs专用血清的IMDM + 0.9%甲基纤维素，同时添加1.5×10-4mol/l硫代甘油、0.28μmol/l抗坏血酸，0.1mmol/l非必需氨基酸、2mmol/l L-谷氨酰胺、1mU/ml TSH、10μg/ml胰岛素及双抗。
1．2．4 进一步向甲状腺细胞诱导分化 将d6 EBs直接接种于铺有0.1%明胶的6孔板及12孔板继续培养11d，培养基用含15% ESCs专用血清的IMDM，添加因子同拟胚体培养，在培养的第13d加入4×10-6mol/l KI。对照诱导组不加TSH、胰岛素和KI。
1．2．5 分化细胞的分析鉴定 于培养第0d、6d、10d，分别用Trizol试剂提取培养细胞总RNA，方法参照试剂说明书。检测Oct4、PAX8、TPO、Tg、TSHR及NIS基因转率水平表达情况，以β-actin为对照。RT-PCR定性分析方法参照ThermoScriptTM RT-PCR System试剂盒说明。以Oligo(dT)20为引物反转录合成第一链cDNA。以各分子的特异引物序列进行PCR，条件为：93℃预变性3min，93℃变性45s、53～65℃（视具体引物）退火30s、72℃延伸45s，进行35 个循环，72℃延伸10min后4℃保存；分别取诱导培养第8d、10d的细胞进行免疫荧光分析。将诱导分化的细胞以PBS洗涤3次，4%多聚甲醛室温固定20min，0.1% Triton X-100 室温渗透7min，山羊血清封闭液处理20min，加入兔抗小鼠一抗，37℃保湿孵育1～1.5h，PBS洗涤3次，加入羊抗兔二抗，37℃保湿孵育，时间视情况而定；诱导培养的第16d胰酶消化6孔板中的贴壁诱导细胞，中止消化后离心重悬，种植于24孔板中，第2d贴壁后进行双色荧光分析。将贴壁细胞以新鲜配制的冷PBS洗涤3次，95％的酒精室温固定10min，0.1% Triton X-100 室温渗透3min，5%牛血清白蛋白（BSA）封闭处理30min；用含1％BSA及鼠抗鼠TTF1和羊抗鼠PAX8一抗混合物的PBS 4度孵育过夜；冷PBS洗涤3次，5min/次；用含1％BSA及兔抗鼠CY3和兔抗羊FITC混合物的PBS室温孵育半h；冷PBS洗涤3次，5min/次；激光共聚焦显微镜观察双色荧光染色。12孔板中的EB诱导细胞直接固定后行双色荧光染色，方法同消化后重悬细胞；电镜观察 诱导培养结束后胰酶消化诱导细胞，中止消化后离心，细胞沉淀经固定、脱水、包埋等处理后行透射电镜观察。取成年昆明种小鼠甲状腺组织进行成体甲状腺细胞(Thyroid Epithelial Cells，TEC)体外培养，培养基为含10%特级胎牛血清的RPMI-1640，添加0.1mmol/l非必需氨基酸、2mmol/l L-谷氨酰胺及双抗。作为形态学对照，显微镜下逐日观察诱导细胞形态学变化。
2 结果
2．1 形态学观察结果 饲养层细胞呈长梭形、多角形、不规则形生长，边界欠清晰。未分化E14小鼠ESCs体积小，为圆形或椭圆形，核大，胞浆少，呈集落、贴壁生长，细胞之间边界不清，排列紧密，集落表面光滑、边缘清晰，偶见少许分化细胞。经悬浮培养后，形成的EBs呈球形，边界清楚，细胞排列紧密，表面凸凹不平。EBs贴壁继续诱导分化，在生长因子作用下，从第8d开始可见贴壁的胚胎体逐渐呈扁平状。中心细胞堆积在一起，形态不清。周边开始出现单层上皮样细胞，边界清晰，呈圆形、类圆形或多角形贴壁生长，与体外培养的昆明小鼠正常甲状腺细胞形态相似。至诱导末期，大部分细胞基本分散成上皮样细胞呈单层贴壁生长，中心剩小部分细胞堆积。
2．2 RT-PCR分析结果 诱导培养第0d即ESCs表达ESCs特有基因转录因子Oct4，不表达甲状腺细胞相关基因PAX8、NIS、TSHR、TPO、Tg等；在培养的第6d和第10d均可检测到诱导细胞中有甲状腺细胞基因PAX8、NIS、TSHR、TPO、Tg的表达而无ESCs特有基因Oct4的表达。
2．3 免疫荧光分析 在培养的第8d可检测到分化细胞中甲状腺细胞分化标记物TSHR、TTF-1强表达，PAX8、TTF-2弱表达；第10d所有甲状腺细胞分化标记物均强表达。阳性细胞均位于胚胎体的周边和表层。作为阳性对照的体外培养的昆明小鼠甲状腺细胞也可检测到TSHR、TTF-1、TTF-2、PAX8等标记物的表达。
2．4 双色荧光分析 对TSH等诱导因子阳性的诱导细胞进行双色荧光检测示，甲状腺细胞分化标记物TTF1和PAX8均可见在部分细胞中表达，激光共聚焦两种荧光的融合图像示部分细胞同时表达TTF1和PAX8，双色荧光和细胞的三种图像融合示荧光标记位于细胞核内。TSH等诱导因子阴性的诱导细胞仅可见TTF1的轻度表达，未见PAX8的明显表达。昆明小鼠甲状腺细胞均匀表达TTF1和PAX8,细胞和双色荧光的融合图像示荧光标记位于细胞核内。
2．5 电镜观察 透射电镜下可见诱导细胞形态多样，部分细胞呈老化显像；可见到内分泌类似细胞，微绒毛、高尔基体明显；但未见细胞内存在分泌颗粒。
3 讨论
目前，在研究ES细胞体外分化时，多将ES细胞用悬浮或悬滴培养法培养，使其聚集形成形似早期胚胎的单个聚集体，称为拟胚体（embryonic body，EB）[2]。它不能完全表现胚胎组织结构，但将它消化成单细胞贴壁培养，并在不同的时段添加不同的培养液或细胞因子等，即可促进细胞分化，形成胚外卵黄囊和胚胎的3个胚层[3]。在EB贴壁培养过程中，可直接分析拟胚体中各种分化的细胞的情况，以筛选出适于某一特定细胞分化方向的培养体系和调控因子。起初人们多用来诱导成造血、神经以及心肌细胞，以后又成功诱导出了骨细胞、胰岛细胞等等。但将胚胎干细胞定向诱导分化成甲状腺细胞的研究很少[4]。
理论上，ES细胞可以分化成各种组织细胞，但是现在“定向分化”的条件还不成熟，诱导分化得到的往往都是包含了多种细胞的混合物，目的分化类型细胞所占的比率不高。实验表明，在干细胞诱导过程中，不管哪种诱导因子，它的作用总是有限的，也不可能诱导出唯一的细胞类型，而且在分子水平的诱导机制仍未搞清，这使在诱导ES细胞分化时带有一定的盲目性。由于甲状腺的发生是需要许多细胞因子参与的复杂过程，其中TSH、insulin或类胰岛素生长因子（IGF-1）、KI等是其发育的必需刺激因子，对甲状腺细胞的发育分化、特异基因的表达及功能的产生等起着重要作用[5]，故本实验模拟甲状腺的体内胚胎发生过程逐步加入以上刺激因子对胚胎体进一步诱导，结果随着EB的成族生长，ES也不断生长，而且细胞在形态学上大多属上皮型细胞，与体外培养的正常甲状腺细胞形态一致。进一步用用荧光免疫检测特异性抗体：包括TSHR抗血清、TTF2和Pax8抗体，在用荧光免疫做特异性标记时，发现在第8d，EB外周出现TSHR阳性细胞，接着相继出现了Pax8和TTF2，我们知道有三种因子：TTF-1、TTF-2和Pax8可以在甲状腺形成的早期表达，并调控甲状腺细胞的分化。其中TTF-2是甲状腺特异性基因表达的负向调节因子[6]，并对TTF-1和Pax8的转录起抑制作用[7]。这些因子的出现提示EB已开始向甲状腺细胞分化。
由于本研究仅从形态学和相关分子水平检测到ESCs定向分化过程中含有甲状腺细胞。然而利用ES细胞诱导甲状腺细胞的研究刚刚起步，在应用于临床以前，需要进一步解决的问题很多：如对于怎样进行精密调控，使ES细胞更精确地向甲状腺细胞定向分化，并具有分泌功能；以及避免ES细胞高度未分化，减少形成畸胎瘤的可能等需要更深入的研究。
本文结果表明，利用胚胎干细胞及拟胚体诱导分化成甲状腺细胞的实验中，诱导细胞中含有甲状腺细胞，且可以表达甲状腺特异性因子，这提示胚胎干细胞可以在体外定向分化成甲状腺细胞，而为今后治疗甲状腺功能减退及临床科研工作提供了实验基础，使胚胎干细胞应用于甲状腺疾病成为可能。
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