【摘要】 目的 探讨下丘脑神经降压素（Neurotensin;NT）降低体温的机制。方法 使用微量注射的方法向核团内注射NT及相应的受体阻断剂及激动剂，观察体温的变化。结果 （1）第三脑室分别注射氟哌啶醇和6-羟多巴胺，均能对抗PO/AH注射NT所致的体温下降（P<005）。（2）腹腔分别注射阿托品和利其丁+心得安，对NT的降温效应无明显影响（P>005）。（3）PO/AH注射NT后，该区的多巴胺含量明显增高（P<005）。（4）注射NT后，该区注射抗NT血清，体温无明显改变（P>005）。（5）改变大鼠所处的环境温度，下丘脑内NT-IR(NT免疫活性物质)含量无显著改变（P>005）。结论 PO/AH外源性NT可能参与体温调节过程，其作用机制可能与该区内多巴胺含量增加有关，并可能通过多巴胺D2受体起作用。生理情况下，内源性NT可能不参与体温调节过程。
【关键词】 大鼠;神经降压素;下丘脑;体温
Research on hypothermy mechanism after injecting neurotensin into preoptic and anterior hypothalamus of rats
ZHU Bao-liangDepartment of Physiology, Jining Medical College, Rizhao 276826，China
【Abstract】 Objective To explore hypothermy mechanism after injecting neurotensin into preoptic and anterior hypothalamus Methods Neurotensin, ANTS, receptor agonist and receptor antagonist were respectively micro-injected into preoptic anterior hypothalamus (PO/AH) and cerebral ventricle of rats The body temperature of the rats was observed Results Aloperidin and 6-hydroxydopamine with IC could antagonize hypothermy of neurotensin (P<005) Atropine, Propranolol and Phentolaminum with IP could not affect hypothermy of neurotensin (P>005) The content of dopamine in PO/AH increased obviously after injecting NT into the area (P<005) Micro-injecting NT into PO/AH after injecting ANTS into the area could not affect the body temperatures of the rats The content of NT in hypothalamus was same in different environment temperature Conclusion The ectogenic NT in PO/AH perhaps has a role in the process of thermoregulation This properly relates to the increasing of content of dopamine and the role due to dopamine D2- receptors is activated Endogenous NT has not a role of thermoregulation
【Key words】 rat; neurotensin; hypothalamus; body temperature
自1973年Carraway等发现神经降压素（Neurotensin;NT）以来，已证明NT是一种脑-肠肽。脑室内和PO/AH注射NT可使实验动物的体温降低，且呈明显的量效依赖关系［1］。由于PO/AH是体温调节的中枢整合部位，且NT在下丘脑的含量很高［2］， 脑室内注射NT引起的体温降低可能是通过PO/AH起作用的，但PO/AH内的NT影响体温调节的机制目前尚不清楚。据此，本文旨在探讨视前区-下丘脑前部(Preoptic anterior hypothalamns;PO/AH)NT影响体温的机制。
1 材料与方法
11 实验分组 实验选用体重为200～250g的健康雄性Wistar大鼠(购自青岛动物所)72只,按以下四个内容随机分为12组(每组6只):(1)注射不同的阻断剂后PO/AH注射NT对体温的影响,PO/AH注射NT 025μg 15min前，第三脑室分别注射利其丁50μg+心得安100μg、阿托品50μg、氟哌啶醇50μg和6-羟多巴胺240μg（均使用生理盐水稀释至300μl），观察体温变化。以第三脑室注射生理盐水300μl 和PO/AH内注射NT025μg作为对照组。 (2)PO/AH注射NT 025μg 1min后，再向该区注射抗NT 2μl，以注射等容量的生理盐水作为对照组。(3)环境温度对下丘脑NT-IR的影响,实验组大鼠分别放置于4℃、22℃和35℃的恒温箱内48h，在此期间每小时更换箱内空气2次，以保持箱内充分的氧气，并给大鼠足够的饮食。迅速断头取脑，置于沸生理盐水中煮5min，取下丘脑称重，匀浆，离心后取上清液。采用放射免疫分析法［4］测定NT-IR的含量。(4)PO/AH注射NT对该区多巴胺含量的影响,实验组大鼠PO/AH注射NT 025μg(2μl)，30min后断头，取脑，浸于冰生理盐水中，取下丘脑称重，荧光分光光度法测定PO/AH多巴胺的含量。PO/AH注射生理盐水2 μl为对照组。
12 实验条件和方法 除“环境因素对下丘脑NT-IR含量影响研究组”外，其余实验组与其对照组大鼠，在实验前后均放置于室温为（2200±10）℃的实验室内，实验动物分别于实验前和实验后的30、60、90和120min测肛温。采用PHILPS肛温计（精确度为001℃），插入直肠内4cm以观察记录体温。动物以20%氨基甲酸乙酯（1g/kg）麻醉后，固定于江湾Ⅰ型C脑立体定位仪上，头部正中切口，剥离骨膜，止血。参照George图谱，用牙科钻在颅骨顶钻孔定位，定位坐标：（1）PO/AH : PO(视前区)：前囟前14mm,旁开10mm；颅骨表面下85mm;AH(下丘脑前部)：前囟后10mm,旁开1mm, 颅骨表面下8mm;(2)第三脑室：前囟后08mm, 旁开1mm, 颅骨表面下40mm。将自制同心套管插入上述部位，套管外径07mm,内管外径04mm,内管前端比外管前端长05mm。以502胶和自凝牙托粉固定同心套管，备作微量注射用。微量注射以1μl/min的速度分别通过套管插入上述部位。注射完毕停留1min后拔出微量注射器。
13 组织学鉴定 每次实验完毕，经套管注射甲苯胺蓝染色，断头取脑，置于10%福尔马林溶液中固定。连续切片，50μm厚，光镜下行组织学鉴定。定位不准确者不记入统计。
14 实验用药物 NT、利其丁、6-羟多巴胺均为美国Sigma公司生产；氟哌啶醇为陕西省医药工业研究所产品；阿托品和心得安系无锡市第四制药厂生产；抗NT血清由青岛大学医学院生理教研室制备，正常兔血清自制。
15 统计学处理 所得数据采用均数±标准差（x±s）表示。实验组和对照组采用两样本均数比较的t检验。取P<005作为判定显著性界限。
2 结果
21 注射不同阻断剂后，PO/AH注射NT对体温的影响 脑室注射阿托品50μg 或利其丁50μg +心得安100μg均不能改变NT引起的体温降低（P>005）（见表1），而脑室注射氟哌啶醇50μg或6-羟多巴胺200μg却能对抗NT的降温效应（P<005）。表1 使用阻断剂后PO/AH注射NT后的体温值
22 PO/AH注射抗NT血清对体温的影响 PO/AH注射抗NT血清（效价为1：14000）和正常兔血清，大鼠体温均无明显改变（P>005）（见表2）。表2 PO/AH注射抗NT血清和正常兔血清后的体温值
23 不同环境温度对下丘脑NT-IR含量的影响 将大鼠分别放置在40℃、20℃和35℃的环境内48h后，断头取脑放射免疫分析法测定下丘脑NT-IR的含量。与对照组相比，大鼠下丘脑内NT-IR的含量无明显改变（P>005）（见图1）。
3 讨论
已证明，脑室内和PO/AH注射NT均可使实验动物的体温降低，且呈明显的量效依赖关系［3］ 。NT在中枢神经系统内以神经递质的方式影响其他神经递质的代谢和释放，例如：NT可直接作用于下丘脑肾上腺素能神经末梢，增加下丘脑肾上腺素的释放量；脑室注射NT可增加某些脑区内乙酰胆碱的代谢，降低乙酰胆碱的含量［4］。为了探讨上述现象是否与NT影响体温有关，本实验在向PO/AH注射NT前15min经腹腔注射利其丁和心得安以阻断肾上腺素能α和β受体，结果表明，上述各阻断剂均不影响NT的降温效应。这表明，NT的降温效应可能与PO/AH内肾上腺素能递质系统和胆碱能递质系统无关。
据文献报道，脑室注射微量的多巴胺，大鼠体温明显降低; 向大鼠的PO/AH注射微量多巴胺，体温也下降明显。NT在下丘脑可增加多巴胺的代谢，且和多巴胺是共存递质［5］。本实验经第三脑室注射氟哌啶醇阻断脑内的多巴胺D2受体、注射6-羟多巴胺耗竭脑内的多巴胺后，均可明显对抗NT的降温作用（P<005）。PO/AH注射NT后30min，断头取脑，下丘脑多巴胺的含量明显增高。提示NT在PO/AH引起体温降低可能与下丘脑内多巴胺的含量增加有关，且通过多巴胺D2受体起作用。故推测脑室注射NT引起的体温降低，可能使NT扩散入PO/AH，并通过增加该区内多巴胺的释放所致。至于NT怎样增加多巴胺的释放。以及多巴胺通过何种途径引起体温变化，目前尚不清楚。有学者认为，NT降低体温是通过降低机体的代谢率实现的。甲状腺切除后的大鼠，体温调节功能部分丧失，脑室注射NT后，体温调节功能障碍加重［6］。但NT是否通过TRH影响甲状腺激素的分泌，目前尚无定论。有资料表明，大鼠脑室注射TRH可对抗NT的降温效应，合适剂量的TRH能完全中和NT的降温效应［7］，但尚没有证据表明PO/AH内的多巴胺递质系统和下丘脑内的TRH有关。
为了探讨PO/AH内源性NT在体温调节中的作用，采用PO/AH注射抗NT血清，结果大鼠体温无明显改变。将大鼠分别放置在4℃、20℃和35℃的环境内48h，断头取脑，下丘脑NT-IR含量也未见明显变化。这提示，在生理情况下，内源性NT可能不参与体温调节过程。
【参考文献】
1 Popp E, Schneider A, Vogel P, et al Time course of the hypothermic response to continuously administered neurotensin Neuropeptides, 2007, 41(5):349-354
2 Reyes R, Valladares F, Díaz-Flores L, et al Immunohistochemical localization of hormones and peptides in the human pituitary cells in a case of hypercortisolism by ACTH secreting microadenoma Histol Histopathol, 2007, 22(7):709-117
3 朱宝亮,程小东,张艳霞,等大鼠下丘脑内神经降压素对体温的影响济宁医学院学报，2004, 27（3）：16-17
4 Belsham DD Hormonal regulation of clonal, immortalized hypothalamic neurons expressing neuropeptides involved in reproduction and feeding Mol Neurobiol, 2007,35(2):182-194
5 Geisler S, Bérod A, Zahm DS,et al Brain neurotensin, psychostimulants, and stress--emphasis on neuroanatomical substrates Peptides, 2008, 27(10):2364-2384
6 Fantegrossi WE, Ko MC, Woods JH, et al Antinociceptive, hypothermic, hypotensive, and reinforcing effects of a novel neurotensin receptor agonist, NT69L, in rhesus monkeys Pharmacol Biochem Behav,2005，80(2):341-349
7 Boules M, McMahon B, Warrington L, et, al Neurotensin analog selective for hypothermia over antinociception and exhibiting atypical neuroleptic-like properties Brain Res, 2001,919(1):1-11
(编辑：悦 铭)
作者单位：276826 山东日照，济宁医学院日照校区生理学教研室
血小板提取液NPY与NT在血液透析中的表达及意义
中国免疫学杂志 1999年第6期第15卷 临床免疫学
作者：罗南萍 杨道理 孟建中① 武翠华 齐法莲 孔立新② 刘文渊①
单位：济南军区总医院免疫科，济南250031
关键词：神经肽Y；神经降压素；血小板；血浆；血液透析
摘 要 目的：探讨血液透析(HD)过程血小板中神经肽Y(NPY)与神经降压素(NT)的含量变化及意义。方法：HD患者35例，对照组30例。采用放射免疫分析法测定血小板提取液和血浆中NPY及NT的含量。结果：与对照组比较，HD患者血小板提取液NPY含量明显降低，血浆中NPY含量明显增高，血小板提取液与血浆NT含量均显著升高。与HD前比较，HD后血浆NT与血小板NPY均明显增高，血小板中NT与血浆NPY明显降低，两者具有显著相关性。结论：在HD过程中，血小板释放的NPY可能是造成肾性高血压和肾血管痉挛的重要病理因素。
中国图书分类号 R446.6
Expression of the platelet extract's neuropeptide and
neurotensin in hemodialysis
LUO Nan-Ping, YANG Dao-Li, MENG Jian-Zhong et al.
Department of Immunology, General Hospital of Jinan Military Region, Jinan 250031
Abstract Objective: To study the changes of neuropeptide Y(NPY) and neurotensin (NT) in platelet extract and plasma in hemodialysis (HD).Methods:There were 35 HD patients and 30 controls.NPY and NT in platelet extract and in plasma were measured by radio-immunity analysis.Results:When compared with control group, NPY was significantly higher in platelet extact and lower in the plasma, while NT was higher both in platelet extract and in plasma in HD patients. When compared with those before HD,NT in plasma and NPY in platelet extract signficantly increased,while NT in platelet extract and NPY in plasma significantly decreased after HD. NPY related to NT significantly.Conclusion:NPY,secreted by platelets, may play an important role in the pathogenesis of renal hypertension and renal vasospasm in HD.
Key words Neuropeptide Y Neurotensin Platelet Plasma Hemodialysis
1987年Ericsson首先发现大鼠等巨核细胞具有合成NPY的能力，并储存于血小板，在血小板聚集时释放［1］。至今对血小板储存、释放NPY和NT的功能以及在人体机能紊乱时的病理生理作用了解甚少。本工作从慢性肾功能衰竭(CRF)时血液透析(HD)患者血浆中分离出血小板，采用放射免疫分析法观察分析了血小板提取液和不含血小板的血浆中NPY与NT的含量变化，探讨血小板释放的生物活性多肽调节血管收缩、舒张活性以及相互拮抗作用对CRF患者的影响。
1 材料与方法
1.1 临床资料 维持性HD患者35例，其中男19例，女16例，平均年龄46.5±5.5岁，均来自本院血液净化中心。原发病分别为慢性肾衰21例、肾小球动脉硬化4例、糖尿病肾病5例、肾盂肾炎3例、多囊肾2例。血透时间为6～74个月(平均39个月)。全部患者内生肌酐清除率Ccr＜10 ml/min，均采用醋酸盐透析液，中空纤维透析器透析膜截留分子量(即允许通过溶质的最大分子量)为12 000。每周透析3次，每次4.0～5.0 h。选择30例健康献血员作为对照组。男17例，女13例，平均年龄为32.5±12.0岁。
1.2 方法
1.2.1 血小板提取液的制备 分别于HD前、后即刻抽取患者静脉血(对照组抽取空腹静脉血)3 ml，立即注入含有40 mmol/L的EDTA2Na 80 μl的试管中，轻轻摇匀，于4℃500 r/min离心30 min后，吸取所有血浆于另一硅化塑料试管中，4℃ 2 000 r/min离心30 min，将上清倾入另一试管中，作为不含血小板的血浆标本，沉淀即为血小板。以生理盐水洗涤沉淀一次，离心倾去上清，于沉淀中加入1 ml生理盐水，充分混匀后，在F800血球计数仪上行血小板计数。调整血小板数为1×109 L-1。将血浆与血小板混悬液置于-30℃以下待测。
1.2.2 样本NPY与NT测定 将血小板混悬液于-30℃～37℃反复冻融3～4次，镜下观察血小板形态大部分裂解、破碎，再以抽干机浓缩抽干后，取1 ml生理盐水复溶，待充分溶解后进行NPY与NT放射免疫测定。人工合成的NPY、NT与相应抗体均为Sigma公司产品(美国进口)。NPY碘标记物为美国Amersham公司产品，125I-NPY稀释后为14 000 cpm/min。125I-NT稀释后为18 000 cpm/min。本抗血清与7种常见多肽类化合物交叉反应小于1%。采用放射免疫非平衡法进行标准曲线及样品和复管检测。此方法两步温育，延长抗原、抗体温育时间，提高方法灵敏度。以上NPY与NT试剂均由解放军总医院东亚免疫技术研究所提供。测量仪器由中国核工业部北京核仪器厂提供的FT-630G微机多探头γ-计数仪。样本浓度按下式计算：
1.2.3 统计学处理 所有数据以(
2 结果
2.1 血小板提取液中NPY与NT水平 与30例正常人比较，HD前NPY水平明显降低，而NT明显增高，均有显著性差异(P＜0.01)。与HD前比较，HD后NPY明显增高，NT则明显降低，并具显著性差异(P＜0.05)，见表1。
表1 血小板提取液及血浆中NPY与NT在HD过程中的表达(
Tab.1 Expressions of NPY and NT in the platelet extract and plasma during HD (
Groups
n
Platelet (ng/109)
Plasma (ng/L)
NPY
P
NT
P
NPY
P
NT
P
Normal
30
58.18±21.29
25.38±13.63
144.06±33.83
70.66±24.72
Pre-HD
35
33.97±19.80
＜0.01
40.47±23.99
＜0.05
423.26±54.97
＜0.01
133.83±92.23
＜0.01
Post-HD
35
65.57±28.571)
＞0.05
15.76±13.22
＜0.05
315.49±50.192)
＜0.01
205.06±134.09
＜0.05
Note:1)compare with pre-HD, P＜0.01；2)compare with pre-HD, P＜0.01
2.2 血浆中NPY与NT水平 与对照组比较，HD前血浆NPY升高极为显著(t=23.36，P＜0.01)。NT水平亦明显升高(P＜0.01)。HD后血浆NPY明显降低，与HD前比较差异十分显著(P＜0.05)，NT则在HD后明显增高(P＜0.05)，见表1。
2.3 样本相关分析 见表2。
表2 血小板提取液和血浆中NPY与NT相关性
Tab.2 The correlation between NPY and NT in the platelet extract and plasma
| Platelet-Plasma | Platelet | Plasma | ||||||||||
| NPY-NPY | NT-NT | NPY-NT |
NPY-NT |
|||||||||
| r | t | P | r | t | P | r | t | p | r | t | p | |
| Pre-HD | -0.67 | 4.08 | ＜0.01 | -0.40 | 2.84 | ＜0.05 | -0.38 | 2.87 | ＜0.05 | 0.68 | 2.98 | ＜0.01 |
| Post-HD | -0.41 | 2.73 | ＜0.05 | -0.18 | 0.88 | ＞0.05 | 0.59 | 2.95 | ＜0.01 | 0.39 | 2.64 | ＜0.05 |
3 讨论
NPY与NT是一组具有拮抗作用的生物活性多肽，它们广泛分布于机体组织内，对机体多种生理活动起重要的调节作用。自1982年发现以来，一直被认为仅存于神经系统中［2］。1987年Ericsson提出血小板中也含有NPY。1991年又进一步证实大鼠血小板中存在与NPY合成相关联的mRNA，表明血小板是合成NPY的主要细胞［3］。本组实验结果发现，在CRF患者血浆分离出血小板提取液中，能测出一定浓度的与NPY、NT特异性抗体相结合的相应抗原，其中NPY水平明显低于对照组，而NT含量则较正常人明显增高。同时检测血浆中NPY与NT的含量均处于高水平状态。这一发现证实了在血小板上可能存在NPY与NT受体，而CRF患者血小板中NPY与NT的代谢紊乱表明血小板不仅参与了凝血机制，可能还参与了肾脏疾病的发病过程。其致病机理可能是由于CRF患者体内毒素聚集作用、凝血机制障碍等原因诱导、活化并释放大量NPY及NT入血浆，致使血浆中NPY与NT浓度增高，这一结果与报道相一致［4］。
结果显示，CRF患者HD后血小板提取液中NPY水平较HD前明显增高，而血浆中NPY水平降低，可能是由于①血液透析作为一种中等强度刺激使病人应激，交感神经处于兴奋状态而使血小板合成释放增加。②HD过程机械性损伤，血小板随血循环与透析膜接触从而被激活的过程。③通过透析患者体内毒素作用减轻，血小板释放入血减少，而血小板中含量大大增加。同时表明透析除能滤出Cr、Bun等有毒物质外，还能清除NPY等中分子多肽类物质，以减少对血管的收缩作用，缓解HD患者以缩血管为主的病理过程。
从相关分析中看出，血小板和血浆中NPY与NT之间关系十分密切，充分证实血浆中大量增加的NPY与NT均来自血小板的释放。有关实验证明，NT的降血压作用是促进肥大细胞释放组织胺等生物活性物质引起［5］。而血小板释放NPY、NT可作为血小板与血管间的一种传递者，直接或与其它缩血管物质(如5-羟色胺等)协同作用，发挥缩血管效应［6］。NPY与NT是一组生物学活性相拮抗的多肽。在HD发病过程中起重要作用。在HD过程增强NT舒血管作用，以拮抗由病理及物理因素造成血小板聚集时NPY大量释放，与5-羟色胺等对血管的强烈收缩作用，是防止HD过程中的严重并发症，进一步提高透析质量，延长患者生命的重要环节。
致谢：感谢解放军总医院李振甲教授给予本研究的指导与帮助。
①济南军区血液净化中心，济南250031
②济南军区总医院检验科，济南250031
作者简介：罗南萍，女，47岁，副主任技师，主要从事神经肽及细胞因子方面的研究
4 参考文献
1 Ericsson A, Schalling M, Mcintyre K R et al. Detection of neuropeptide Y and its mRNA in megakaryocytes: Enhanced levels in artain autoimmune mice. Propc Natl Acad Sci USA,1987;84:5585
2 Ailen J M, Bloom S R. Neuropeptide Y: a putative neurotramsmitter. Neurochem,1986;8:8
3 Ericsson A, Hemsen A, Lundberg J M et al. Detection of neuropeptide Y -Like immunoreactivity and messenger RNA in rat platelets: The effects of vinblastine reserpine, and dexamethasone on NPY expession in bloold cells. Exp Cell Res,1991;192:604
4 Myets A K, Farhat M Y, Vaz C A et al. Releasa of immunoreactive-neuropeptide by rat platelets. Bionchem Biophys Res Commun,1988;155:158
5 蔡 强.神经降压素.国外医学*消化系统疾病分册，1986；6(3)：129
6 杨耀芳，杨丽华，施广璞.大鼠血小板中的神经肽Y及其对血管收缩的影响.生理学报，1993；45(4)：400
〔收稿1998-02-18 修回1998-11-29〕
National Centre for Microbiology—National Institute of Health Carlos III, 28220 Majadahonda (Madrid), Spain
ABSTRACT
More than 50% of the nontypeable (NT) pneumococcal strains received in our laboratory for reference purposes are isolated in sporadic cases of conjunctivitis. To determine the genetic structure of the population of these NT conjunctival strains, we analyzed 75 pneumococci (40 NT and 35 typeable) isolated from conjunctivas and 30 (15 NT and 15 typeable) isolated from other sources. The NT and typeable conjunctival strains grouped in separate clusters, whereas NT and typeable pneumococci isolated from other sources were similarly distributed. NT conjunctival strains belonged to two well-differentiated clonal lineages. The first, represented by three newly described sequence types, featured fully antibiotic susceptible strains and appeared to be characteristic of conjunctival tissue; the second, represented by the previously described ST344, had a pattern of multiresistance to penicillin, tetracycline, and erythromycin and shared a genetic background with some NT strains isolated from other sources.
INTRODUCTION
Streptococcus pneumoniae is an important pathogenic bacterium associated with pneumonia, septicemia, meningitis, and otitis. It is also a common cause of acute conjunctivitis, particularly in children, but also in adults (11, 19). For reference purposes, our laboratory receives many pneumococcus samples from all origins that have been isolated in Spanish hospitals (8). Around one-third of the strains isolated from children below the age of 6 months that were studied in our laboratory between 1990 and 1999 caused acute conjunctivitis (9).
Pneumococcal serotyping usually fails to detect a small number of strains that do not react with antipneumococcal typing sera. Nontypeable (NT) strains are infrequently isolated from sterile clinical specimens (2.2%), in which case they are rarely implicated as causes of invasive disease (2, 12), since they are otherwise relatively common in nonsterile samples (10%). The identification of these NT pneumococci is dubious (14, 17), particularly in nonsterile specimens, and they may be confused with other Streptococcus species. The association between the presence of NT isolates and the occurrence of conjunctivitis was first suggested in 1977 (10) in a retrospective study of the incidence of capsular types in a Boston hospital between 1935 and 1974.
Further studies associated NT strains with outbreak and sporadic cases of conjunctivitis (1, 6, 18, 20), and a recent report has confirmed NT S. pneumoniae-like strains isolated from an outbreak of epidemic conjunctivitis as being S. pneumoniae (3).
In the last 10 years, approximately 50% of the NT pneumococcal strains received in our laboratory have been isolated from cases of conjunctivitis, and the frequency of these noncapsular strains was five times that found in other pathologies (laboratory data).
In general, the NT strains have been characterized in cases related to outbreaks, but as yet we have little information concerning those strains isolated from sporadic cases.
The purpose of this study was to characterize NT pneumococcal strains isolated from conjunctivas in sporadic cases of conjunctivitis in Spain between 1997 and 2002. The overall objective was to determine whether the population genetic structure of this group of strains was similar to that found in typeable strains isolated from conjunctivas. In addition, the genetic relatedness of NT isolates from conjunctivas and NT strains from other origins was also analyzed. Pulsed-field gel electrophoresis (PFGE) (15) and multilocus sequence typing (MLST) (5) molecular markers were used for these pneumococcal strains.
MATERIALS AND METHODS
Strains. (i) Identification and typing. A total of 14,650 pneumococcal isolates were received in our laboratory between 1997 and 2002. Of these, 1,068 strains were isolated from conjunctivas.
All isolates were identified as S. pneumoniae by their distinctive colony morphology on sheep blood agar and were confirmed by the optochin susceptibility and bile solubility tests.
Serotyping was initially performed with a dot blot assay as previously described (7), using 46 anticapsular sera provided by the Statens Serum Institut (Copenhagen, Denmark). Strains that gave uncertain results with this technique were typed by the Quellung reaction. Strains that did not react at all with any sera were classified as NT.
All NT strains were confirmed as S. pneumoniae by using the AccuProbe S. pneumoniae identification kit (Gene Probe, Inc., San Diego, Calif.) (4).
(ii) Strains analyzed by PFGE. Thirty-five NT and 25 typeable strains isolated from conjunctivas of different geographical origins within Spain between 1997 and 2000 were chosen at random to be analyzed by PFGE.
(iii) Strains analyzed by MLST. Sixty pneumococcal strains were analyzed by MLST. Fifteen of these (5 NT and 10 typeable strains) represented the most frequent profiles obtained by PFGE analysis. Another 15 (10 NT and 5 typeable strains) were selected from the conjunctival strains isolated in 2001 and 2002. Finally, 30 strains (15 NT and 15 typeable) were selected from isolates recovered from other sources during the same period.
PFGE. Genomic DNA was prepared in agarose blocks as previously described (13) and digested with SmaI (MBI Fermentans, Quimigranel, Spain) in the recommended restriction buffer. PFGE was performed in 1% agarose MP (Roche Diagnostics Corporation, Indianapolis, Ind.) in 0.5x Tris-borate-EDTA buffer at 12°C and 6 V/cm in a CHEF-DR II (Bio-Rad Laboratories, Hercules, Calif.) for 22 h with a switching time of 0.1 to 40 s.
Band patterns were compared by using the Molecular Analyst Fingerprint (Bio-Rad Laboratories). A dendrogram was generated from the data by the unweighted-pair group method using average linkages, with the Dice coefficient and a tolerance of 1% (see Fig. 2).
MLST. Briefly, internal fragments of the aroE, gdh, gki, recP, spi, xpt, and ddl genes were amplified and then sequenced in each direction with primers described by Enright and Spratt (5). The sequences of each of the seven loci were compared with those of all the known alleles, by using the programs available on the MLST website (http://www.mlst.net/) to assign allele numbers. The same software was used to define the allelic profile and sequence type (ST) of each isolate. Data were analyzed, and the appropriate dendrogram was generated (see Fig. 3), with a sequence type analysis and recombinational test.
RESULTS
The conjunctival strains represented 7.2% of all pneumococcal isolates studied between 1997 and 2002. This percentage is similar to that found for otic strains (6.9%), while invasive isolates (i.e., isolates from blood or cerebrospinal fluid) recovered in the same period represented 45%.
NT strains were the most frequent (23.2%) of the conjunctival isolates. This NT percentage is higher than those found for strains isolated from other sources (Fig. 1). The most representative serotypes of the pneumococci causing conjunctivitis in Spain during the period 1997 to 2002 were 19A/F (14.1%), 6B (9.8%), 23F (8.8%), 14 (5.3%), 6A (4.6%), 23A/D (3.7%), and 11 (3.3%). This distribution does not differ significantly from that of serotypes found among pneumococci isolated from cases of invasive disease.
PFGE analysis. Digestion of DNA with SmaI yielded a total of 39 different band pattern profiles. Nineteen of the 35 (54.3%) NT strains from conjunctivas corresponded to only four closely related profiles, while the 25 typeable strains corresponded to 22 profiles (Fig. 2).
In general, the isolates were distributed in clusters in accordance with their typeability. NT isolates appeared in two clusters, A and C (Fig. 2), and the typeable pneumococci were grouped in cluster B.
MLST typing analysis. The relationships based on the allelic profiles of the 60 pneumococcal isolates analyzed by MLST are shown in Fig. 3. Thirty-three STs were found. Nevertheless, all of the NT strains isolated from conjunctivas belonged to only four STs, three of which (ST941, ST942, and ST943) were closely related. The pneumococci grouped in these three related STs belonged to PFGE cluster A (Fig. 2), and their allelic profiles shared six of the seven alleles analyzed. However, these strains shared only two or three of their alleles with the fourth profile (ST344) (Fig. 3).
The NT pneumococcal strains isolated from other sources were mainly distributed in other STs (Fig. 3) in the dendrogram. It was particularly striking that the three related STs corresponded solely to NT strains isolated from conjunctivas, while ST344 grouped not only NT pneumococci isolated from conjunctivas but also other NT strains isolated from different sources. Likewise, typeable strains isolated from conjunctivas had no particular distribution pattern, appearing in different STs with typeable strains isolated from other sources.
DISCUSSION
Sporadic conjunctivitis has traditionally been associated with different microorganisms (11), including typeable pneumococcal strains (10). However, NT pneumococcal strains have been proposed to be an important cause of infection, particularly in outbreaks of conjunctivitis (18, 20). Nontypeability may result from a loss of capsular material due to specific but poorly understood mechanisms (20). In fact, to express little or no capsular polysaccharide can be advantageous in that it allows the colonization of several tissues, including the conjunctival surface (1, 21).
In this study, a population of NT pneumococcal strains isolated from conjunctivas in sporadic cases was analyzed by comparing it with other populations of strains (typeable and NT) isolated from conjunctivas and other sources. Between 20.5 and 28.7% of the conjunctival pneumococci studied in our laboratory were NT (Fig. 1), a frequency similar to that described by other authors (10, 20). This contrasts with the low percentage (2.2%) of NT strains isolated from other sources (2) and with the infrequent appearance of NT strains in invasive diseases (12).
According to the data obtained by PFGE (Fig. 2), NT conjunctival strains were distributed in two separate clusters, A and C, which were well differentiated from that of the typeable isolates of the same origin. This suggests that NT strains isolated from conjunctivas may possess particular genetic characteristics suggesting a clonal nature.
On the basis of MLST, NT strains from conjunctivas again formed a group separate not only from the typeable strains isolated from conjunctivas but also from typeable strains of other origins, thereby yielding two distinct and well-differentiated clonal lineages, one made up of STs 941, 942, and 943, and one consisting of ST344 (Fig. 3). However, most of the patterns found in NT strains isolated from other sources were grouped with typeable isolates and had common or closely related STs. These may be considered pneumococci with similar genetic backgrounds that express little or no capsular polysaccharide.
The population structure of the typeable pneumococci isolated from eyes is similar to that found in all pneumococcal strains (13). Throughout the dendrogram, typeable strains isolated from eyes and from other sources were grouped within the same clonal lineages. Some typeable pneumococcal strains are thus able to survive in conjunctivas and give rise to sporadic cases of conjunctivitis. NT strains of STs 941, 942, 943, and 344 might be better adapted to the colonization of eye tissue. However, nontypeability alone cannot explain the specific location of these pneumococcal strains in the eyes, since most of the NT pneumococci isolated from other origins did not belong to these two clonal lineages. The isolates belonging to ST344 (Fig. 3) showed a typical pattern of multiresistance to penicillin, tetracycline, and erythromycin, while the other NT isolates (ST941, -942, and -943), grouped in the other cluster, were all susceptible. ST344 falls within cluster C by PFGE. Two strains with this ST, isolated from blood, have been described in the MLST database previously: one had the multiresistant pattern, and the other was resistant to penicillin but not to erythromycin. Therefore, NT ST344 pneumococci might not represent a clonal lineage exclusive to the conjunctiva. In fact, in our study, some NT strains from other sources were found in this ST. To address this matter, a large number of strains of this specific ST need to be analyzed.
By contrast, the other NT pneumococci isolated from the eye, which grouped together in three closely related STs, corresponded to cluster A and might represent a characteristic conjunctival-tissue cluster. However, other authors have not found specific clusters among NT strains isolated from sporadic cases of conjunctivitis (1). In the previous study, BOX-PCR was used to characterize the isolates; this methodology might not reflect the genetic structure of the pneumococcal population analyzed as well as MLST does.
The NT strains belonging to the two clonal lineages described in this study might represent Spanish endemic pneumococcal clones involved in conjunctivitis cases over a long period. In fact, several other NT pneumococci isolated from conjunctivas in Spain many years before, in the 1980s, also belong to the same clonal lineages (laboratory data). Two previous studies have shown that closely related NT strains were responsible for conjunctivitis outbreaks in different geographical areas over at least 20 years (6, 16).
Further studies including a larger number of NT pneumococcal strains isolated from conjunctivas and other tissues from different and widely separated countries are required to confirm our findings. Otherwise, we cannot discount the possibility that these NT pneumococci represent a group genetically divergent from typical pneumococcal strains. Additional studies with these strains, including the analysis of the capsular operon but also some other genes, might better clarify their real phylogenetic position.
ACKNOWLEDGMENTS
This work was supported by The Spanish Pneumococcal Infection Study Network, Red Temática de Investigacion Cooperativa (G03/103), Ministerio de Sanidad.
We thank the laboratories that sent us the pneumococcal strains.
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Departments of Laboratory Medicine and Pathology and Pediatrics, University of Minnesota Medical School, Minneapolis, Minnesota
Department of Obstetrics, Gynecology, and Reproductive Sciences, Magee Women's Hospital and University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
Departments of Pediatrics and Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas
ABSTRACT
Group B streptococci (GBS) are serotyped according to capsular polysaccharide (CPS) type (Ia to VIII); an isolate is classified as nontypeable (NT) if no detectable CPS is found. Surface-localized protein antigens (, , R1, and R4) serve as additional markers to classify GBS isolates, which is particularly useful since NT isolates often express one or more of these proteins. To compare genetic resemblance among isolates with similar protein profiles, we studied 58 NT isolates digested with the SmaI macrorestriction enzyme prior to pulsed-field gel electrophoresis (PFGE). Of these 58, 15.5% expressed only, 20.7% expressed +, 15.5% expressed R4, and 25.8% expressed R1,R4, while 22.4% of the isolates expressed no detectable proteins. The largest PFGE profile group, with 48% of the isolates, was group 4, composed primarily of isolates that expressed R1,R4 or no proteins. The second most common profiles were 3 and 32, each with 13.8% of the isolates. Since NT isolates in profile group 4 were highly related to type V isolates, as demonstrated by PFGE profiles, we investigated 45 type V isolates. Two-thirds of the type V isolates within profile group 4 were classified into subgroup 4a, compared to 28.2% of 39 NT isolates. Only 11% of the V/R1,R4 isolates were identical to the prototype group 4 profile, in contrast to 75% of the NT/R1,R4 isolates. A shift of type V isolates into profile 4 subgroups may be indicative of a genetic change over time. PFGE is a valuable approach for comparison of GBS isolate relatedness and for monitoring of NT and typeable GBS isolates for potential clonal divergence.
INTRODUCTION
Group B streptococcal (GBS; Streptococcus agalactiae) isolates are classified according to their capsular polysaccharide (CPS) into one of nine types: Ia, Ib, and II to VIII (13, 19). However, when tested by routine typing methods, approximately 2.9% of colonizing isolates and 1.4% of invasive isolates lack a detectable CPS type and consequently are categorized as nontypeable (NT) (4, 9, 10).
In addition to the CPS type, protein markers can be useful for classification since most GBS isolates express either the c or the R surface-localized protein(s) (9, 10). The c protein is made of two components that are distinguished on the basis of their reaction with trypsin; is trypsin resistant, while is trypsin sensitive and binds immunoglobulin A. Isolates may possess only one or both of these components (14). The R proteins (R1, R2, R3, and R4) are the second group of surface proteins and are trypsin resistant; isolates may express one or more of them (10, 18).
Identification of surface-localized proteins is an important aspect of the classification of GBS isolates since some proteins are highly associated with specific CPS types. For example, protein is commonly expressed by the majority of serotype Ia isolates, while more than half of serotype V isolates possess the R1 and R4 proteins (10). Thus, these CPS type-protein profile associations may permit one to compare similarities among NT and typeable isolates on the basis of their protein profiles.
Because pulsed-field gel electrophoresis (PFGE) is highly reproducible and yields well-resolved bacterial DNA, it is commonly used in molecular and epidemiological laboratories to classify and type bacterial isolates (2, 16). Using PFGE, our laboratory previously classified 78 NT GBS isolates into DNA profile groups on the basis of their DNA macrorestriction patterns (4). To continue validation of PFGE as an effective tool for classifying NT isolates, we recently studied 58 additional NT isolates. In addition, we investigated further the genetic relatedness between NT/R1,R4 and V/R1,R4 isolates and examined their DNA macrorestriction profiles for evidence of genomic divergence.
MATERIALS AND METHODS
Bacterial isolates. All NT and type V isolates were received in our laboratory between 1998 and 2002 from a multicenter collaborative study (Pittsburgh, Houston, and Seattle). The 58 NT GBS clinical isolates studied included 1 invasive and 57 colonizing (34 vaginal, 23 rectal) isolates; the colonizing isolates were from 35 nonpregnant women. Most (n = 50) of the isolates were from Pittsburgh, while 7 were from Seattle and 1 was from Houston. The 45 type V GBS clinical isolates studied included 4 invasive and 41 colonizing (22 vaginal, 19 rectal) isolates; the colonizing isolates were from 39 nonpregnant women. Most (n = 42) of the isolates were from Pittsburgh, while 3 were from Houston.
Growth conditions. GBS isolates were stored frozen at –30°C in Todd-Hewitt broth (Difco Laboratories, Detroit, Mich.) with 2% sheep blood until studied. They were grown overnight in Todd-Hewitt broth for CPS and protein typing and on 5% sheep blood agar plates (Remel, Lenexa, Kans.) for 2 days at 37°C for molecular studies (3).
Serotyping. To determine the CPS type and surface-localized protein profile, isolates were typed by HCl extraction and Ouchterlony double immunodiffusion in agarose as previously described (10, 14). When necessary, the HCl extract was concentrated to enhance weak precipitin reactions and/or grown in broth with increased glucose and buffer to enhance CPS production (1). Isolates without detectable CPS were further analyzed by PFGE.
Molecular analysis by PFGE. A rapid PFGE assay was performed on isolates with no detectable CPS in accordance with our published protocol (3), a modification of the method of Fasola et al. (8). Briefly, bacteria were embedded in agarose plugs and then treated with mutanolysin and proteinase K to lyse the cell wall and precipitate the protein. The bacterial DNA was digested in situ with the infrequently cutting restriction enzyme SmaI before the DNA fragments were resolved by PFGE. NT isolates with similar protein profiles were assembled into groups and studied together with the PFGE prototype typeable isolate for that particular protein. Each gel also contained a lambda DNA ladder (Bio-Rad) and our internal control, strain 89-022 (Ib/+).
DNA macrorestriction band pattern analysis. For digital analyses, the gel was photographed under UV light with a DC 40 digital camera (Kodak, Rochester, N.Y.) and the image was imported into the 1D digital imaging software (Kodak) to gather information about the migration, intensity, size, and quantity of DNA bands from each isolate. Visual comparison of the PFGE profiles of the isolates to the DNA profiles of the prototypes was done by using our modification of the criteria of Tenover et al. (16). To achieve more precise analyses, we created the following method to compare the typeable and NT isolates for genotypic relatedness on the basis of their band patterns (4). Each DNA profile group had a prototype to which all similar isolates were compared. An isolate that did not have a band pattern identical to that of the prototype was assigned a letter to designate a profile subgroup. An isolate with a one- or two-band difference from the prototype DNA profile was assigned the letter a, one with a three-band difference was assigned the letter b, one with a four-band difference was assigned the letter c, and one with a five-band difference was assigned the letter d. Isolates with five or more band differences were not considered to be related to the PFGE profile group and were assigned a different profile number. Classification of isolates into PFGE profile subgroups ranging from a to d allowed one to identify immediately to what extent an isolate was related to the prototype. However, it was possible for isolates within a specific subgroup to have nonidentical band patterns, since band differences were defined as either the presence or absence of a band compared to the profile prototype.
RESULTS
Nontypeable GBS isolates. A comprehensive summary of both protein expression patterns and PFGE profile data is shown in Table 1. The most prevalent protein profile among the 58 NT isolates was R1,R4, with 15 isolates (25.9%); the least common protein profiles were R4 and only, each with 9 isolates (15.5%). Thus, 21 isolates expressed c protein (36.2%) and 24 isolates expressed R proteins (41.4%), while 13 isolates (22.4%) did not express a detectable protein.
Analysis of the PFGE band profile patterns resulted in assignment of the 58 NT isolates to 15 of 35 DNA macrorestriction profile groups. Among these 35 groups were profiles 32 to 35, which we added recently to enable classification of an additional five isolates. As shown in Table 1, 9 -only isolates were distributed among six different PFGE profiles (1, 11, 13, 15, 16, and 33), 12 + isolates were distributed among five PFGE profiles (2, 19, 32, 34, and 35), and 9 R4 isolates were distributed among four PFGE profiles (3, 4, 22, and 28), while all 15 R1,R4 NT isolates were classified into DNA profile group 4. Of 13 isolates lacking a detectable protein, 12 were classified into profile 4, while 1 isolate was assigned to profile group 16.
Profile 4 was the most prevalent PFGE group, with 28 isolates (48.3%), and included isolates with three different protein profiles: 15 R1,R4 isolates, 1 R4 isolate, and 13 isolates with no detectable protein (Fig. 1). Profiles 3 and 32, each with five isolates, were the second most common PFGE profiles. All isolates within newly created profile 32 were + and included the only invasive NT isolate. All PFGE profile groups with fewer than three isolates were consolidated into a miscellaneous group for Fig. 1. This group of NT isolates was composed of two isolates each from PFGE profile groups 2, 15, 16, and 28 and one isolate each from groups 11, 13, 22, 33, 34, and 35.
Clonal divergence of type V isolates. Since almost half of the 58 NT isolates were classified within profile group 4 and the prototype for this profile group is a V/R1,R4 isolate, it seemed warranted to study V/R1,R4 isolates more extensively and to compare their PFGE band patterns with those of the NT isolates in this profile group. Of the 84 PFGE profile group 4 isolates used in this analysis (Table 2), 45 were type V and 39 were NT (15 R1,R4 isolates and 13 isolates without protein from the 58 NT isolates were included, plus an additional 11 from the same time period).
Three representative gels are shown in Fig. 2, with NT and V isolates compared to the profile group 4 prototype in lane 9 of each panel. Panel A shows the high degree of homogeneity found in NT/R1,R4 isolates, with six of the seven isolates displayed in this gel classified into profile group 4 and only one (lane 7) classified into subgroup 4a. In contrast, the V/R1,R4 isolates in panel B show some genetic divergence from the classic V/R1,R4 profile group 4 prototype (lane 9), since more than half of the isolates in this gel were classified into subgroup 4a. Likewise, the PFGE profiles of the isolates in panel C were representative of recent V/R1,R4 isolates and all were profiled as subgroup 4a or 4b. The markedly different restriction pattern in lane 8 of panel C was that of a type VII/R1,R4 isolate that was classified into profile group 17.
The distribution of the NT and serotype V isolates within PFGE profile group 4 or its subgroups is summarized in Table 2. Whereas 22 (56.4%) of the 39 NT isolates had profiles that were identical to that of the profile 4 prototype, only 8 (17.8%) of the 45 serotype V isolates were classified into profile 4. When the isolates were analyzed by their protein profiles, it was found that 18 (75.0%) of the 24 NT/R1,R4 isolates were identical to the prototype isolate of profile group 4, as shown in Fig. 2A. However, in contrast to the NT/R1,R4 isolates, the NT/none group was heterogeneous, with only 4 (28.6%) isolates classified into profile 4 and the remaining 10 of the 14 isolates classified into subgroups 4a to 4d. Similar analysis of the typeable isolates showed that the majority (88.9%) of the V/R1,R4 isolates were in profile subgroup 4a (18 isolates) or 4b (6 isolates). The same trend was observed with V/R4 and V/none isolates, with the majority in subgroup 4a rather than group 4.
DISCUSSION
This work not only verified the value of grouping isolates according to expressed surface proteins in studying NT isolates but also reaffirmed the validity of classifying NT GBS isolates according to DNA macrorestriction PFGE profiles. Furthermore, since we analyzed all of the isolates from a 5-year period, the results of this epidemiological study will assist researchers in monitoring GBS trends with regard to the serotyping of NT isolates and monitoring divergence among type V GBS isolates.
The importance of continuous monitoring of NT GBS isolates by PFGE was emphasized by our discovery of PFGE band patterns not seen previously in our laboratory. In 2002, we reported that 135 isolates were classified into 26 different PFGE profile groups (4); currently, our laboratory has identified 35 profiles. These additional, unique band profile groups will permit the classification of more isolates for future reference.
The distribution of NT isolates expressing the same protein profile among PFGE profile groups was consistent with trends that we have observed previously with typeable GBS isolates. We had reported that isolates of the historical serotypes (Ia, Ib, II, and III) were distributed among a number of PFGE band patterns (8), whereas most isolates of the more recently emerging serotypes were in a very limited number of profile groups, as exemplified by our finding that all of the type V isolates studied were classified into one PFGE profile group (4). In addition, we found that certain protein profiles were highly associated with specific CPS types, such as with type Ia, + with type Ib, R4 with type III, and R1,R4 with type V (10). We observed similar trends in our NT isolates. Specifically, NT isolates with an protein profile such as only that would be associated with serotype Ia were distributed among more profile groups than the NT/R1,R4 isolates, which, like the V/R1,R4 isolates, were all in the same profile group, reflecting perhaps less genetic diversity (7).
It was of interest that NT isolates classified into PFGE profile group 4 or its subgroups, and therefore considered to be highly related to V/R1,R4 isolates, accounted for 48.3%, nearly half, of the NT isolates studied. This reflected a slight increase since 2002, when we found that of 78 NT isolates, 30 (38.5%) were in this profile group (4). We do not know why profile group 4 constituted such a significant percentage of the NT isolates. One can speculate that it may reflect their advantage in colonization or perhaps that poorly encapsulated or nonencapsulated variants or variants related to type V were more likely to be produced than by isolates of other serotypes. PFGE profile group 4, described here, is identical to PFGE subtype O, described by Elliott et al. (7), which made up the majority of isolates of GBS type V from 1986 to 1996.
Among typeable GBS isolates, whether invasive or colonizing, CPS type V has increased in prominence over the past 15 years. In the early 1990s, the percentage of type V isolates causing invasive disease in all patient groups increased from approximately 3% to about 20% (5, 7) and to 29% for nonpregnant adults (12). We reported that type V was responsible for 14% of invasive infections in neonates and for 22.6% of those in pregnant women (19) and that this serotype accounted for approximately 12% of the colonizing GBS isolates from neonates or parturient women at the time of delivery (13). However, the percentage may be even higher since in a study of vaginal and rectal colonization in 102 nonpregnant women, we found that if multiple colonies were picked from the initial culture plate for serotyping, the relative percentage of type V isolates was 14.3%, versus 13.6% when only one colony was processed, as is customary in most laboratories (10, 11). Furthermore, our recent data from a much larger number of women with paired vaginal and rectal cultures taken at up to four culture visits, indicated that 18.4% of the women were colonized with type V, underscoring the importance of this CPS type (unpublished observations).
Analyses of the V/R1,R4 isolates suggested a genotypic change since the majority of recent isolates were classified as having profile 4a, unlike our past isolates that had a PFGE band pattern indistinguishable from that of the prototype isolate for profile group 4. Genetic diversity among type V isolates has also been observed by Thomas-Bories et al. (17), as they found 11 distinct patterns in 64 isolates. In contrast to our V/R1,R4 isolates, the NT/R1,R4 isolates appeared to have retained genetic homogeneity, with three-quarters still classified within PFGE profile 4. This change in the distribution of NT and type V isolates among subgroups of profile 4 was of interest, since our previous publication demonstrated that most of the NT and type V isolates producing proteins R1 and R4 or no proteins were in profile group 4 (4).
Because of recent interest in vaccine development for prevention of GBS disease, precise identification of GBS isolates is of importance. Consequently, DNA dot blot hybridization and PCR methods have been developed as an alternative to conventional serotyping and have proven to be highly sensitive techniques (6, 15). However, PFGE is an excellent adjunct to these methods since the analysis of DNA band patterns enables one to compare isolates and assess the degree of relatedness among them, providing an overall view of the extent of homogeneity or clonal divergence within groups of isolates over time.
ACKNOWLEDGMENTS
This study was supported in part by contract N01-AI-75326 from the National Institutes of Health, Bethesda, Md.
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［摘要］ 目的 观察硝普钠，多巴胺与利尿剂合用治疗低血压合并慢性充血性心力衰竭（心衰）的疗效及安全性，及其对患者血浆氨基末端脑钠肽（NtproBNP）浓度的影响。方法 将心功能Ⅲ～Ⅳ级的105例心衰患者，随机分为治疗组和对照组，对照组给予利尿剂与氨茶碱治疗；治疗组在对照组基础上加用硝普钠与多巴胺，治疗观察10天，并对两组的心功能改善评价、症状改善及心脏彩超进行对比，并采用ELISA法对两组患者治疗前后血浆NtproBNP进行测定。结果 治疗组总有效率，显效率，治疗后左室射血分数（LVEF）显著高于对照组（P<0.05）；治疗组治疗后血浆NtproBNP水平显著低于对照组（P<0.05）。结论 硝普钠与多巴胺联用在治疗低血压合并慢性充血性心衰中有重要意义，血浆NtproBNP水平在心衰治疗疗效评价中有较高敏感性。
［关键词］ 心力衰竭；硝普钠；多巴胺；氨基末端脑钠肽
Effects of combination of sodium nitroprusside， dopaine in the treatment of chronic congestive heart failure and effects on plasma NtproBNP level
HE Qin， ZHANG Liyun， CHEN Manhua.
Cardiovascular Department， Wuhan Central Hospital，Wuhan 430015， China
［Abstract］ Objective To detect the effect and safety of combination of sodium nitroprusside， dopamine in the treatment of chronic heart failure， and the effect on the level of plasma NtproBNP.Methods 105 patients with heart failure of 3～4 degree of heart function were divided into treatment group and control group randomly.The control group was treated with diuretic and aminophylline.And the treatment group was treated with sodium nitroprusside， dopaine additionally.The treatment was last 10 days.Comparison was done between the two groups， as the evaluation of improvement of heart function， improvement of signs of heart failure，the change of left ventricular ejection fraction (LVEF) and left ventricular enddiastolic diameter（LVDd） by ultrasonography of heart as the evaluation index， and the level of plasma NtproBNP was measured before and after the treatment in each group.Results The total utility rate，total of significant utility rate，left ventricular ejection fraction (LVEF) in treatment group were significantly higher than those in control group after treatment（P<0.05）.And the plasma NtproBNP level was significantly lower than that in control group (P>0.05).Conclusion Effects of combination of sodium nitroprusside， dopaine in the treatment of low blood pressure with chronic congective heart failure was significant.Plasma NtproBNP level has considerably high sensibility in the evaluation of treatment effect of heart failure.
［Key words］ heart failure;sodium nitroprusside;dopaine;Ntpro brain natriuretic peptide
低血压合并慢性充血性心衰患者病情危重，一般抗心衰治疗效果不佳，现利用多巴胺合并硝普钠，再加用利尿剂、氨茶碱治疗此类心衰，以症状、心脏多普勒超声检查及血浆氨基末端前脑钠肽(Ntpro brain natriuretic peptide， Nt-proBNP)为治疗效果检测指标，观察其治疗效果及安全性，报告如下。
1 资料与方法
1.1 一般资料 入选病例为2005年3月～2006年2月住院的105例低血压合并心力衰竭（心衰）患者，所有患者血压水平为(100~75)/(65~40)mmHg，心功能评价Ⅲ～Ⅳ级以纽约心脏病协会（NYHA）提出的心衰分级法分级：治疗组55例，男35例，女20例，平均年龄67.5岁，其中急性心肌梗死患者23例，缺血性心肌病患者10例，肺心病患者5例，扩张型心肌病患者6例，风湿性心脏病患者11例；对照组50例，男27例，女23例，平均年龄66.9岁，其中急性心肌梗死患者21例，缺血性心肌病患者11例，肺心病患者3例，扩心病患者7例，风湿性心脏病患者8例，两组患者均排除肝肾功能不全等其他并发症。
1.2 方法 全部病例均接受氨茶碱与利尿剂治疗，给药剂量氨茶碱针0.125 mg，速尿针60 mg加入100 ml生理盐水中静脉滴注。治疗组患者除接受氨茶碱与利尿剂治疗外，还每日以硝普钠针12.5 mg，多巴胺针20 mg加入生理盐水50 ml中静脉泵入（5 ml/h）治疗心衰，治疗10天后评价治疗效果。
1.3 疗效判定标准 （1）按临床心功能改善评价［1］，显效：心功能改善2级；有效：心功能改善1级；无效：心功能未达到有效标准者；（2）心衰症状改善，治疗前后记录两组患者心衰症状（胸闷、气急），体征（呼吸、血压、心率、发绀、两肺啰音、双下肢中度水肿）；（3）心脏彩超记录两组治疗前后左室舒张末期内径（LVDd），左室射血分数（LVEF）。（4）以NtproBNP ELISA试剂盒测定两组患者治疗前后血浆NtproBNP水平。
1.4 统计学方法 对两组病例治疗前后呼吸、心率、血压、血浆NtproBNP水平进行t检验，对心功能改善总有效率、显效率、心衰症状缓解率进行χ2检验。
2 结果
2.1 两组患者的心功能改善情况的比较 治疗组总有效45例，显效7例，无效3例，对照组总有效31例，显效9例，无效10例。治疗组显效率81.8%(45/55)，总有效率94.5%(52/55)；对照组显效率62.0%（31/50），总有效率80.0%（40/50）。经χ2检验方法对两组患者的显效率及总有效率分别作统计学分析，治疗组与对照组相比有显著性差异（P<0.05）。
2.2 疗效比较 两组患者治疗前LVDd、LVEF、心率、血浆NtproBNP水平及心衰阳性症状体征与对照组治疗前相比差异无显著性，经治疗后LVDd、LVEF、心率与血浆NtproBNP水平较之治疗前均有显著改善，其中治疗组治疗后LVEF及血浆NtproBNP水平较治疗前改善更为显著（P<0.01），较对照组治疗后差异也有显著性（P<0.05），见表1。
表1 两组治疗前、后症状、超声心动图改变 (略)
注：与同组治疗前相比，*P<0.05，**P<0.01；与对照组治疗后相比，△P<0.05
2.3 不良反应 两组患者治疗中及治疗后均未出现低血压，肾功能受损等不良反应。
3 讨论
多巴胺主要兴奋β受体，也具有一定兴奋α受体的作用，小剂量2～5 μg/（kg·mm），可使肾、肠系膜、冠状动脉和脑血管扩张，尤其是改善肾血流量，提高肾小球滤过率及利尿作用，还可作用于支气管平滑肌，解除痉挛，改善通气。硝普钠是临床上常用的降压药，能选择性松弛血管平滑肌，对动静脉的作用相等，引起血管扩张，外周阻力下降，肺动脉及右心房压也下降，特别对心功能不全患者，可使肺毛细血管楔压和右房压降低，降低心脏的前后负荷，改善心功能，使每搏量和心排血量显著增加。因此，这两种药物在心衰的治疗中均有其重要意义。
严重的心衰患者其心脏前后负荷均增加，造成心脏舒缩功能障碍，同时，由于神经体液因素的参与，肾素-血管紧张素-醛固酮系统激活，使外周血管阻力增加，交感神经进一步兴奋，儿茶酚胺增多，心率增快，水钠潴留加重，使心脏的前后负荷增加，导致心衰加重。
给予心衰患者利尿剂与氨茶碱的治疗，其意义主要在于减轻心脏前负荷，改善肺水肿及支气管痉挛。但是此法对于严重的心衰患者治疗效果往往不甚满意，尤其是心衰合并低血压患者，其肾动脉血流充盈不足，利尿效果较差，且由于其血压低，临床上应用利尿剂时亦受到限制。此时在利尿剂治疗的基础上加用多巴胺、硝普钠有望改善心衰的各种病理生理过程，有利于心衰的纠正。本研究中，合用多巴胺与硝普钠治疗的治疗组患者治疗后LVDd、LVEF、心率与血浆NtproBNP水平较之治疗前均有显著改善，其中治疗组治疗后LVEF及血浆NtproBNP水平较对照组治疗后差异也有显著性（P<0.05），显示出良好的效果。
脑钠肽(BNP)在心衰的诊断、预后及治疗上均有重要意义，尤其在判断心衰的严重程度及预后的价值已得到国内外学者的认同［2， 3］。NtproBNP是心室肌细胞合成BNP的同时，等摩尔生成的一段无生理活性的肽段，其血浆稳定性高于BNP，半衰期也相对较长，测定较为方便准确，因此本研究采用测定血浆NtproBNP在治疗前后的水平变化作为疗效评定指标之一。研究结果发现，硝普钠、多巴胺治疗低血压合并慢性心衰患者较单用利尿剂、氨茶碱疗效显著，治疗组治疗前血浆NtproBNP水平与对照组相比差异无显著性，而治疗10天后其水平明显较对照组低，这充分说明了硝普钠、多巴胺在治疗中的意义，也说明血浆NtproBNP在评价心衰程度及治疗疗效中较高的敏感性。
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(编辑：唐 城)
作者单位： 430015 湖北武汉，武汉市中心医院心内科
【摘要】 目的 探讨联合检测5’-NT、ADA、GGT在肝细胞性黄疸和阻塞性黄疸鉴别诊断中的价值。方法 对本院40例健康人群、35例急性黄疸型肝炎和21例阻塞性黄疸病人的血清进行了5’-NT、ADA、GGT、ALT、ALP五种酶活性平行检测，并做对比分析。结果 5’-NT、GGT结果在急性黄疸型肝炎组、梗阻性黄疸组中均有非常显著增高，且其阳性率均为100%。而ADA在急性黄疸型肝炎组中虽只有88.5%阳性率，但其在梗阻性黄疸组中有非常低的阳性率，在两病例组中差异有非常显著性。结论 5’-NT、ADA、GGT的联合检测是诊断梗阻性黄疸的良好指标，若3项指标均增高，则为肝细胞性黄疸；若5’-NT、GGT显著增高，而ADA正常则为梗阻性黄疸。
【关键词】 5’-核苷酸酶；腺苷脱氨酶（ADA）；γ-谷氨酰基转移酶（GGT）；黄疸
【Abstract】 Objective Discussing measure 5’-NT, ADA, GGT jointly in liver cellular jaundice and obstructive jaundice on the differential diagnosis.Methods To 40 healthy crowds of our hospital, 35 cases acute jaundice type hepatitis and 21 cases obstructive jaundice, the patients serum go on 5 ’-NT, ADA, GGT, ALT, ALP five kinds enzyme activity parallel measured, make compare and analyse. Results 5 ’-NT, GGT result in acute icterohepatitis group and in obstructive jaundice has extremely remarkably increased, its positive rates are 100%. But ADA in acute icterohepatitis group although only then 88.5% positive rates, but it has the extremely low positive rates in obstructive jaundice group, has the extremely remarkable difference in two illness groups. Conclusion 5’-NT, ADA, GGT union examination is diagnoses obstructive jaundice the good targets, if three targets raises, then for liver cellular jaundice; If 5’-NT, GGT remarkably raises, but ADA is normal then for obstructive jaundice.
【Key words】 5’-NT；ADA；GGT； jaundice
肝细胞性黄疸与阻塞性黄疸的鉴别在临床和实验室都较困难，由于阻塞性黄疸中部分病人有轻度的肝损害，而肝细胞黄疸中部分病人有肝内胆管阻塞，故尚难找到特异的鉴别两类黄疸的方法。近年来，血清5’-核苷酸酶（5’-NT）、腺苷脱氨酶（ADA）测定在肝胆疾病中的应用多见于报道。对本院40例健康人群、35例急性黄疸型肝炎和21例阻塞性黄疸病人的血清进行了5’-NT、ADA、r-谷氨酰基转移酶（GGT）等多种酶活性检测，并做对比分析，以探讨联合检测5’-NT、ADA、GGT在急性黄疸型肝炎和阻塞性黄疸鉴别诊断中的价值。
1 资料与方法
1.1 对象与分组 正常对照组：选择本院体检健康（通过物理检查、影像学检查、实验室检查排除肝脏疾病）人群40例作为对照组，其中男21例，女19例；年龄18～58岁，平均48.3岁。急性黄疸型肝炎组：35例，系本院传染科住院确诊病例，临床诊断标准参照2000年全国病毒性肝炎学术会议修订方案，其中男24例，女11例；年龄16～51岁，平均47.8岁。梗阻性黄疸组21例， 均经临床确诊并施行外科手术成功的病人， 其病因: 胆石症16例，胆总管癌及壶腹部肿瘤各1例，胰头癌3例。其中男15例，女6例；年龄19～ 66岁， 平均51.4岁。
1.2 观测指标 5’-核苷酸酶、腺苷脱氨酶（ADA）、γ-谷氨酰基转移酶（GGT）、血清丙氨酸氨基转移酶（ALT）、碱性磷酸酶（ALP）。
1.3 测定方法 血清ADA试剂（宁波美康生物科技有限公司）；ALT、ＡＬＰ、GGT（上海科华）；5’-ＮＴ（法国Biomerienx公司）。以上都采用酶促反应连续监测速率法。
1.4 测定仪器 日立-7170全自动生化分析仪、光栅分光光度计。按说明书设置参数。
1.5 统计学方法 统计软件采用SPSS10.0系统。各组数据采用t检验进行统计学分析，各组间计量资料以x±s表示；以P＜0.05为差异有显著性。以5’-ＮＴ、ＡＤＡ、ＧＧＴ、ＡＬＴ、ＡLP测定结果正常值上限作为阳性界值。正常值: 5’-ＮＴ≤9u/L、ＡＤＡ<25u/Ｌ、ＧＧＴ<47u/Ｌ、ＡＬＴ<40u/Ｌ、ＡLP<135u/L。
2 结果
各组各项目观测指标的测定结果及阳性率见表1，表2。
表1－表2 （略）
从表1可见5’-NT、GGT、ALT、ALP四指标在急性黄疸型肝炎组及梗阻性黄疸组中的结果均值均明显高于正常对照组，差别均具有显著性（P＜0.05）；ADA在急性黄疸型肝炎组中结果均值明显高于正常对照组，差异具有显著性（P＜0.05），但其在梗阻性黄疸组中的结果均值与正常对照组相近，差别无显著性（P＞0.05）。
急性黄疸型肝炎组中各指标与正常对照组相比只有ALT增幅最大，最大可达10倍左右，其余4指标均只有中等程度增高。梗阻性黄疸组中各指标与正常对照组相比5’-NT、GGT、ALP 3项指标均有较大增幅，增幅达4～5倍，并呈平行关系；ALT在梗阻性黄疸组中只有轻度增高，甚至少数病例结果出现正常；ADA在梗阻性黄疸组中基本相近，差异无显著性。
从表2可见急性黄疸型肝炎组各指标中5’-NT、GGT、ALT 3项指标阳性率均为100%，ADA、ALP两指标阳性率稍低，分别为88.5%、85.7%。梗阻性黄疸组各指标中5’-NT、GGT、ALP阳性率最高，均为100%；其次为ALT，阳性率为66.7%；ADA最低，阳性率仅为14.2%。
3 讨论
肝脏是具有复杂功能的重要器官，其具有很强的代偿能力。肝功能生化实验中，每一个检查指标都只能反映肝功能的某一个侧面，且变化的特异性受多种因素影响，在各种肝胆疾病以及疾病发展的不同阶段，由于代谢障碍引起的各种酶学指标变化都会不同，因此将反映肝胆系统损伤的各种指示酶组合在一起进行检测，有助于临床获得更直接、快捷和准确的诊断信息。
5’-ＮＴ在肝脏中主要存在于毛细胆管膜、窦状膜及枯否细胞的细胞膜上，在肝胆系统发生病变时，其释放的5’-ＮＴ经胆汁酸处理，去垢后释放入血，使血清中5’-ＮＴ活性增高，故其在胆道疾病中的升高比在肝脏疾病中显著［1］。ＡＬＰ、ＧＧＴ一般作为传统胆汁郁积标记酶应用于临床诊断。本文显示梗阻性黄疸组中各指标与正常对照组相比5’-NT、GGT、ALP 3项指标均有较大增幅，增幅达4～5倍，并呈平行关系。血清中的5’-ＮＴ则主要来源于肝胆系统，因此它对肝胆系统疾病的诊断具有高度的特异性［2］，而血清中ＡＬＰ来源广泛，且与骨化过程密切相关，在很多骨骼疾病时明显增高；另外，血清中ＧＧＴ可受到药物（苯巴比妥类、苯妥英钠）、酒精的影响。本文结果表明血清5’-ＮＴ是诊断鉴别急性黄疸型肝炎和阻塞性黄疸的一个较敏感的实验室指标。
ＡＤＡ是嘌呤核苷分解代谢过程中起关键作用的酶，广泛分布于人体各器官中，正常人血清中含量低于25u/Ｌ，肝内ＡＤＡ 90%存在于胞浆中，其余位于核内，与核酸代谢有关。当肝细胞损伤坏死或膜通透性增加时，肝细胞内ＡＤＡ释放于血中引起血清ＡＤＡ活性增加，所以ＡＤＡ测定可作为反映肝实质损伤的指标。据报道ＡＤＡ测定有助于阻塞性黄疸和肝细胞黄疸的鉴别［3］，前者明显低于后者，肝细胞黄疸明显升高，本文结果与之相符。
ALT作为一个传统的主要肝功能检测项目在临床应用非常广泛。本文结果显示，在急性黄疸型肝炎组中ＡＬＴ最为敏感，与文献报道一致［4］。但在梗阻性黄疸组中只有轻度增高，甚至少数病例结果出现正常，说明ALT作为胆汁淤积标记酶敏感性较低，并且与其他各组病例有一定重叠。
笔者将5’-NT、ADA、GGT 3项指标组合起来进行研究，以期探讨它在急性黄疸型肝炎和阻塞性黄疸鉴别诊断中的价值。结果表明，5’-NT、GGT在急性黄疸型肝炎组、梗阻性黄疸组中均有非常显著增高，且其阳性率均为100%。而ADA在急性黄疸型肝炎组中虽只有88.5%阳性率，但其在梗阻性黄疸组中有非常低的阳性率，在两病例组中差异有非常显著性。故本文认为5’-NT、ADA、GGT的联合检测是诊断梗阻性黄疸的良好指标，若3项指标均增高，则为肝细胞性黄疸；若5’-NT、GGT显著增高，而ADA正常则为梗阻性黄疸。
【参考文献】
1 林其燧，文庆成主译. 临床化学诊断方法大全. 北京∶北京大学出版社，1990，945-952.
2 Sunderman FW Jr. The clinical biochemistry of 5’-nucleotidase. Annclin Lab Sci，1990，20（2）:123-139.
3 戴万荣，郭金星.检验医学临床应用.上海:上海科学普及出版社，2001，122-123.
4 黄承乐. 十一种生化项目在肝脏疾病中变化的研究. 右江民族学院学报，1999，21（5）:763-765.
作者单位：422000 湖南邵阳，邵阳市中心医院检验科
（编辑：齐 永）
【摘要】 目的 检测各型肝胆疾病、骨骼疾病患者血清5′-核苷酸酶（5′-NT），谷丙转氨酶（ALT）和γ-谷氨酰转肽酶（γ-GGT），碱性磷酸酶（ALP）酶活性的变化，观察在肝胆疾病和骨骼疾病中的临床意义，同时与正常健康者作对照。 方法 用法国Biomerieux公司（5′-NT）诊断试剂盒测定血清5′-NT酶活性，同时测定患者血清谷丙转氨酶（ALT）和γ-谷氨酰转肽酶（γ-GGT），碱性磷酸酶（ALP）酶活性的变化。 结果 发现在肝胆疾病和骨骼疾病时，血清5′-NT酶活性升高，尤其是急性黄疸型肝炎、肝癌、胆道疾病患者血清5′-NT酶活性差异有非常显著性（P<0.01）时，随病情好转，血清5′-NT酶活性有所降低。 结论 （1）血清5′-核苷酸酶（5′-NT）酶活性测定对诊断肝胆疾病、骨骼疾病具有重要价值。（2）联合检测血清5′-核苷酸酶（5′-NT），谷丙转氨酶（ALT）和γ-谷氨酰转肽酶（γ-GGT），碱性磷酸酶（ALP）酶活性的检测有助于鉴别诊断肝脏疾病、胆道疾病和骨骼疾病。
【关键词】 5′-核苷酸酶;谷丙转氨酶;γ-谷氨酰转肽酶;碱性磷酸酶;肝胆疾病;骨骼疾病
Clinical significance of assaying serum5′-NT，ALP，ALT，γ-GGT in hepatic and gall diseases，osteal diseases
LI Shun-jun，HUANGWen-fang，RAO Shao-qin.
Department of Clinical Laboratory，the People's Hospital of Sichuan Province，Chengdu610072，China
【Abstract】 Objective To study the serum active enzymatic of5′-NT and ALT，γ-GGT and ALP and in hepatic and gall diseases，osteal diseases，compared with the normal period as the control group.Methods The serum active enzymatic of5′-NT were examined using Biomerieux sa Francais Enzyline5′-NT kit，together with ALT，γ-GGT and ALP.Results It showed that the serum active enzymatic of5′-NT in hepatic and gall diseases，osteal diseases levels were increase，especially the more significant increase of the serum active enzymatic of5′-NT in level ap-peared in the patients with hepatic and gall diseases，osteal diseases.It showed that the application of5′-NT in acute hepatitis，live cancer and osteal diseases were significantly higher than that of controls.The serum active enzymatic of5′-NT may be useful in diagnosis and differentiation of some hepatocirrhosis and liver cancer.With the diseases get-ting better，the application of5′-NT gradually decrease.Conclusion （1）The serum active enzymatic of5′-NT level was significantly correlated with types and in hepatic and gall diseases，osteal diseases.（2）serially observed the serum active enzymatic of5′-NT changing in process of hepatic diseases might be a biological prognostic marker，to-gether with ALT，γ-GGT and ALP，it can be used to distinguish and diagnose most hepatic and osteal diseases.
【Key words】 5′-nucleotidase;hepatic and gall diseases;osteal diseases;ALP;ALT;γ-GGT
血清5′-核苷酸酶（5′-Nucleotidase，5′-NT），是一种特殊的磷酸水解酶，主要分布于心、肝、脑、肌肉、肾和肺，在肝内分布于胆小管、肝窦和Kupffev细胞中，血清5′-核苷酸酶（5′-NT）活性升高主要用于肝胆疾病诊断及肝胆疾病与骨骼疾病的鉴别诊断 ［1］ 。我们对205例肝胆、骨骼疾病患者及62例正常健康者分别测其血清5′-核苷酸酶活性，并针对性选择具有诊断意义的几个肝功能酶ALP、ALT、γ-GGT测定，所得结果作如下探讨。
1 材料与方法
1.1 研究对象 205例均为我院住院确诊病人，男120例，女85例，年龄10～80岁。其中急性黄疸性肝炎38例，慢性肝炎26例，酒精性肝炎16例，肝硬化51例（失代偿期23例、代偿期28例），肝癌14例，胆道疾病40例，骨骼疾病20例;62例正常健康者作对照，均为我院正常体检者。肝病患者诊断均符合2000年9月西安全国第十次病毒性肝炎及肝病学术会议方案的诊断标准 ［2］ 。
1.2 检测方法
1.2.1 血清5′-核苷酸酶测定 用法国Biomerieux公司（5′-NT）诊断试剂盒，操作按说明书进行。
1.2.2 血清ALP、ALT、γ-GGT测定 按常规方法进行测定。
1.3 仪器 AU-2700全自动生化分析仪。
1.4 统计学处理 各组结果用t检验完成，正常参考值0～11U/L。
2 结果
对205例肝胆、骨骼疾病患者及62例对照组分别测定5′-NT、ALP、ALT、γ-GGT结果见表1。表1 不同人群血清5′-NT、ALP、ALT、γ-GGT活性测定结果 （略）注:各组与正常对照组比较， * P<0.01， # P<0.05
2.1 血清5′-NT酶活性检测 由表1可看出，肝胆疾病患者血清5′-NT酶活性与对照组比较均明显升高，尤其是急性黄疸型肝炎、肝癌、胆道疾病患者血清5′-NT酶活性差异有非常显著性（P<0.01），慢性肝炎、酒精性肝炎、肝硬化失代偿患者与对照组比较，亦差异有显著性（P<0.05），各型肝病患者随病情的好转，恢复期或代偿期时，血清5′-NT酶活性有所降低，逐渐恢复正常，例如对39例住院治疗的急性黄疸性肝炎患者血清5′-NT酶活性的动态监测，结果发现:当进入缓解期时，血清5′-NT酶活性经统计学处理结果为（8.2±1.3）U/L，呈下降趋势;对23例肝硬化失代偿患者血清5′-NT酶活性的动态监测，结果发现:当肝硬化失代偿期转变为代偿期时，血清5′-NT酶活性经统计学处理结果为（8.6±1.5）U/L，也呈下降趋势。但在骨骼疾病患者血清5′-NT酶活性正常。
2.2 血清ALP酶活性检测 急性黄疸型肝炎、慢性肝炎、酒精性肝炎、肝硬化失代偿期、肝癌、胆道疾病、骨骼疾病患者与对照组比较，血清ALP酶活性均有升高（P<0.05），差异有显著性，尤其是胆道疾病、骨骼疾病患者血清ALP酶活性差异有非常显著性（P<0.01）。
2.3 血清ALT、γ-GGT酶活性检测 由表1可看出，急性黄疸型肝炎、酒精性肝炎、肝硬化失代偿期、肝癌、胆道疾病患者血清ALT、γ-GGT酶活性升高，急性黄疸型肝炎血清ALT酶活性差异有非常显著性（P<0.01），慢性肝炎、酒精性肝炎、肝硬化失代偿期和肝癌患者血清γ-GGT酶活性有升高，差异有非常显著性（P<0.01）。
3 讨论
肝病包括肝实质细胞损害，梗阻性疾病及二者的并发症，在肝实质性病变中，为监测活动性肝细胞损害常用的试验是血清酶活性的测定，有人提出多种肝功能实验的组合 ［3］ 。5′-核苷酸酶（5′-Nucleotidase，5′-NT）是一种特殊的磷酸水解酶，主要分布于心、肝、脑、肌肉、肾和肺，在肝内分布于胆小管、肝窦和Kupffev细胞中，在肝胆系统发生病变时，其释放的5′-核苷酸酶（5′-NT）经胆汁酸处理发生去垢后释放入血，使血清5′-核苷酸酶活性升高，因此主要用于肝胆疾病的诊断。同时监测血清肝功能实验的其它组合酶的活性，能鉴别诊断肝胆疾病与骨骼疾病，为临床提供重要的诊断价值 ［4，5］ 。
急性黄疸型肝炎患者，由于肝细胞大量受损，肝功能障碍，血清转氨酶水平明显升高，血清5′-核苷酸酶酶活性升 高，呈正相关，因此，联合检测血清5′-核苷酸酶酶活性和ALT有助于急性黄疸型肝炎早期诊断。
慢性肝炎、酒精性肝炎患者，由于肝细胞仍大量受损，肝功能较差，血清肝功能酶ALP、ALT、γ-GGT都升高，血清5′-NT也升高，但γ-GGT升高较为显著，因此联合监测血清肝功能酶ALP、ALT、γ-GGT和5′-NT酶的活性为慢性肝炎、酒精性肝炎患者的治疗提供重要依据。肝硬化患者失代偿型，肝功能仍较差，肝功能血清酶学升高，而血清5′-NT也升高。对于代偿型，随着肝功能的好转，血清5′-NT活性下降，因此，血清5′-NT酶活性也可作为肝硬化患者肝脏储备功能及预后判断的指标。
肝癌患者血清5′-NT活性显著升高，有资料报道γ-GGT也是一个很敏感的非特异肝癌标记物，本文检测结果也说明这一点，因此联合检测，可为肝细胞癌、肝硬化和肝炎的鉴别诊断提供重要的依据 ［6］ 。
胆道疾病患者，由于胆道阻塞，胆管上皮细胞损害和胆汁淤积，使血清5′-NT、ALP酶活性明显升高，与阻塞的严重程度呈正相关，联合监测有利于胆道疾病的早期诊断。而骨骼疾病患者，只是血清ALP酶活性明显升高，而血清5′-NT酶活性正常，因此可用于胆道疾病和骨骼疾病的鉴别诊断。在60例正常健康对照组中，血清5′-NT酶活性与性别、年龄无明显差异。
综上所述，各型肝病患者检测肝功能的同时，检测血清5′-核苷酸酶酶活性有非常重要意义，可为临床早期诊断和鉴别诊断提供重要依据，可作为肝病预后判断参考指标。
【参考文献】
1 陈伟，杨沛.5′-核苷酸酶的测定及临床意义.中华医学检验杂志，2004，27（3）:154-155.
2 全国第十次病毒性肝炎及肝病学术会议.中华传染病杂志，2001，19（1）:53.
3 关春杰.肝病实验室诊断浅谈.检验医学技术通讯，2001，3（3）:14-15.
4 吴倩，曾视伦.5′-NT的自动化测定及临床价值探讨.重庆医科大学学报，2003，23（3）:375-378.
5 叶应妩，王毓三.全国临床检验操作规程.第二版.南京:东南大学出版社，1997，231-232.
6 邱红，朱月蓉.5′-核苷酸酶和甲胎蛋白的联合检测对原发性肝癌的诊断意义.南京医科大学学报，2004，24（6）:644-646.
作者单位:610072四川成都，四川省人民医院检验科
（编辑:悦 铭）
1 NT的化学本质与合成释放
1973年，Carraway等 ［1］ 在分离牛的下丘脑P物质（Subˉstance P，SP）时，发现一种新肽，能引起麻醉大鼠裸露皮肤的血管明显扩张，因其来自神经系统，具有降低血管张力的作用，故命名为神经降压素（Neurotensin，NT）。1975年，有学者 ［2］ 运用Merrifield固相法首次合成NT，证实其由13个氨基酸残基组成，分子量是1673。N末端缺少游离的-NH2，C末端可被羧肽酶（Carboxypeptidase）水解，其排列顺序如下:焦谷-亮-酪-谷-门酰-赖-脯-精-精-脯-酪-异亮-亮-OH。NT分子的后6个氨基酸残基具有其全部生物活性，即NT（8-13）（精 8 -精 9 -脯 10 -酪 11 -异亮 12 -亮 13 ），但NT（8-13）并不是NT在体内产生的降解片段之一。改变NT分子某个氨基酸，人工合成的NT（8-13）类似物有Eisai复合物 ［3］ 、NT64L、NT66L、NT67L和NT69L，均可以通过血-脑屏障。
1987年，Dobner PR等 ［4］ 成功克隆NT基因。人们进一步发现:NT与和它结构相似的，由6个氨基酸残基组成的神经介素（Neuromedin N，NN）（H-赖-异亮-脯-酪-异亮-亮-OH），均来自哺乳动物的脑和肠道的同一大分子前体（Precursor，pro-NT/NN），它们均位于该前体的羧基端，中间被成对的碱性氨基酸赖-精相隔，前体的中间部分存在另一个蛋白编码区，生成NN样肽（NN-likesequence）。NT和NN是内切蛋白酶（Endoproteases）作用于同一前体不同切点的产物。此前体在大脑，主要产生中枢NT和NN;在肠道，主要分解出周围NT和羧基端以NN结尾的大分子肽—大NN（LargeNN）;在肾上腺，产生NT、大NN和羧基端以NT结尾的大分子肽—大NT（Large NT） ［5］ 。研究表明 ［6］ ，中枢NT和NN是在脑组织去极化时释放。和其它神经肽一样，在分泌细胞中，NT既可以单独存在于直径为100nm的大囊泡内，释放于神经末梢突触结构部位;也可以与5-HT、DA、NE等共存于直径为50nm的小囊泡内，与这些经典神经递质同时释放，对其分泌、传递信息等发挥调节作用 ［6］ 。在血脑屏障的薄弱部位中枢NT可能进入血液循环 ［7］ 。胃肠道内周围NT和大NN是在摄食后被释放入血液循环系统。
2 NT的分布、代谢及受体
NT在中枢及外周神经系统中广泛、非均匀分布，不同动物之间差异较大。在中枢神经系统中，NT广泛存在于脑组织的神经细胞、纤维和末梢 ［8］ 。人脑中的NT主要分布于下丘脑、正中隆起、垂体柄、黑质、脑干结构（包括导水管周围灰质）、蓝斑、中缝核和上下丘之间 ［9］ ，在与精神心理活动有关的下丘脑的杏仁核、弓状核、边缘系统中的分布尤为密集 ［10，11］ ;其次在大脑皮质（钩回）、海马、基底神经节、腺垂体、扁桃体和丘脑;三叉神经、视神经和齿状核的分布最少。在胃肠道，NT分布在消化道粘膜的特异性内分泌细胞—N细胞中，以远端小肠为主 ［8］ 。NT在心脏主要分布于窦房结、房室结、心房、心室和乳头肌。此外，还分布于肾上腺髓质、甲状腺及癌组织中。用放免法测定不同性质的抗血清所检测的NT片段及所得的血浆值不同。从目前有限的文献报道中初步确定血浆中的NT浓度为20～60pmol/L。NT的生物半衰期约40s，其降解和失活机制现已明确 ［5］ 。在血液循环和组织中，NT被作用羧基端的金属内切肽酶（Metalloendopeptidases）缓慢灭活，主要降解部位是精 8 -精 9 ，于脯 10 -酪 11 及脯 7 -精 8三处肽键。NN被氨肽酶迅速灭活;大NN的降解机制还不明确。
1990年，Nakanishi等 ［12］ 克隆出第一个NT受体NTS1（旧称NTR-1），并证明它属于G蛋白偶联受体家族（the family of G protein-coupledreceptors）。主要分布于黑质、中脑腹侧背盖区等处。几年后，Gully D等 ［13］ 合成首个非肽类的NT受体拮抗物SR48692，Rostene W等 ［14］ 证实它能阻断一部分NT的中枢和外周效应，但不能抑制中枢注射NT导致的镇痛和降低体温作用。第二代的拮抗物SR142948比SR48692的作用强大，Gully D等 ［15］ 证明它可以抑制脑室注射NT导致的镇痛和降低体温作用。近年，又克隆出了两种新的NT受体:一种是类似于NTSR1的nts2，仍属于G蛋白偶联受体超家族，分布于嗅觉系统、大脑和小脑皮层、海马和某些下丘脑核;nts3从结构上完全不同于NTSR1和nts2 ［16］ ，分布于脑、脊髓、心脏、骨骼肌、胎盘和睾丸，其生理作用尚不明确。在各系统各组织中的3种受体，其亲合力有高低两种类型，亲合常数分别为0.5nmol/L和9nmol/L，最大结合容量分别为41fmol/mg和100fmol/mg蛋白。在与靶细胞受体结合时，主要是其碳末端4个氨基酸侧链与受体疏水集团相互作用的结果。当NT和受体结合后，可以激活腺苷酸环化酶通过cAMP、Ca 2+ 等作
为第二信使而发挥生物效应。
3 NT与神经系统
在中枢神经系统，NT具有显著的镇痛、调节多巴胺神经传导、降低体温和刺激腺垂体激素分泌的作用。并因此和某些疾病的发病有关。
3.1 镇痛作用 NT在脑中分布广泛，包括与痛觉有关的脑区。脑池或脑室注射极微量NT，可减弱动物对痛刺激的反应，而外周注射则不出现。NT的镇痛作用稍强于内啡肽，而比吗啡和脑啡肽强千倍。陈家津等 ［17］ 报道了功能性头痛患者脑脊液NT含量变化，比对照组减少了78.91%;头痛不发作时NT比治疗前头痛发作增加53.89%，但仍比正常人减少67.54%。其中12例患者进行追踪，经药物治疗头痛症状消失后，再次抽取脑脊液测定NT的含量时，发现NT的含量均比治疗前头痛发作时升高，但仍低于对照组。
3.2 NT与情感障碍 从上世纪70、80年代开始，Pert CB等 ［18］ 提出，神经多肽及其受体构成了受心理影响的精神身体网络，参与脑与身体之间的脑、腺体、免疫系统的交流网络，并可能是情感机制的生化基础。有学者曾注意到未经治疗的精神分裂症患者脑脊液中NT含量低，治疗后可回升至正常 ［19］ 。但Weiss DW等 ［20］ 研究表明，以色列居民在海湾战争中及战后焦虑程度高于战前，同时血浆NT水平升高。已有的研究 ［21］ 表明，NT可能在多巴胺能系统功能中占有一席之地。Cechetto DF等 ［22］ 研究表明，应激诱导的心血管功能不良的皮质定位可能在岛叶，因为岛叶接受来自内脏的神经冲动信息，而其生化机制在于丘脑杏仁核中核中NT及神经肽Y的介导。
3.3 调节激素分泌 NT参与调节哺乳动物的神经内分泌系统，经用TRH阻断镇静剂及NT效应证实:NT对前垂体、下丘脑下部的激素分泌具有很强的调节作用，主要作用于丘脑下部水平。应用RIA和免疫组化技术发现，NT样免疫活性物质分布于下丘脑和前垂体，在中央隆起也有储存和释放 ［23，28］ ，这证明:NT参与神经内分泌调节由其分布决定。脑内注入NT能抑制黄体生成素的分泌，增加生长激素分泌。外周注入NT也能使垂体生长激素、催乳素、促甲状腺素分泌增加。Bozzola M等 ［24］ 报道，血浆NT水平是生长激素抑制激素受抑制的外
围标志之一。
3.4 降低体温 脑室内或颅内静脉窦中注入微量的NT，即可使啮齿动物的体温下降，而外周注入NT则无作用。降温的程度与NT的剂量和动物所处环境温度有关。
3.5 NT与神经系统疾病 李晨等 ［25］ 测定了脊髓病变脑脊液13例中，四肢锥体束征6例，其NT含量比对照组增加14.1倍。而7例脊髓病变性质待诊与对照组相比无明显变化。有学者曾注意到未经治疗的精神分裂症患者脑脊液中NT含量低，治疗后可回升至正常 ［19］ 。在老年痴呆病脑脊液神经肽含量变化来看，阿尔茨海姆病患者脑脊液中精氨酸加压素、β-内啡肽、生长抑素和NT含量比对照组均有显著降低。随着神经肽的基础和临床研究的不断深入。也将使人们对老年痴呆病机制会有更多的了解，并为临床治疗奠定基础。
临床测定血浆和脑脊液中NT水平，在研究脑血管疾病方面资料较多 ［24～27］ ，结果显示:出血性脑血管病患者脑脊液NT含量比正常人增加，其原因可能是（1）脑部某些疾病因素影响，脑内NT合成增加;（2）脑组织受疾病因素的破坏时，脑组织中的NT释放并进入脑脊液;（3）可能与出血时脑脊液含血有关。脑血栓形成所致的缺血性脑血管病患者脑脊液的NT含量比对照组减少，而引起脑内NT合成减少，释放入脑脊液的NT量降低，导致患者脑脊液的NT含量明显减少。因此，出血性脑血管病和缺血性患者脑脊液NT含量呈现显著的反向变化，这对两类不同脑血管病临床的鉴别诊断及治疗措施等方面提供有益的参考。从急性缺血性脑血管病患者血浆NT含量变化来看:发病后2周内血浆NT含量明显低于正常对照组，并随着病情好转，而NT 水平恢复正常。血浆NT水平逐渐恢复者均无意识障碍及死亡，结果显示与预后有关。由于血浆较脑脊液获取容易，便于动态观察，故血浆NT测定为脑血管病患者提供一个有潜力的临床生化指标。
总之，NT作为多巴胺传导和前垂体激素分泌的神经调质，在大脑中发挥降低体温和镇痛的作用，并与精神疾病的情绪的变化有关 ［28］ 。NT还有许多生理效应无法解释，特别是与多巴胺系统的交互作用原理，因此可能还存在许多未知的NT受体亚型。而且NT类似物作为治疗用药的前景也是值得探讨的。
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作者单位:1 100039北京市中西医结合医院神经内科
2 410078中南大学湘雅医院
（收稿日期:2004-08-21） （编辑田 雨）
