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1、 II摘 要 肝硬化(hepatic cirrhosis)是由多种原因引起的肝脏损害，表现为肝细胞变性坏死，纤维组织增生，肝细胞结节状再生，肝小叶结构变形，血液循环途径被改建，最终导致肝硬化。根据病因分类，肝硬化主要有病毒性肝硬化、酒精性肝硬化、胆汁性肝硬化、自身免疫性肝硬化及隐原性肝硬化等。我国以病毒性肝硬化为主，其中主要为乙型肝炎病毒感染造成，很多病人无法长期规范化治疗最终致肝硬化。肝硬化病人有门脉高压和肝功能障碍，病人常因消化道大出血和肝性脑病死亡。因此，肝硬化治疗是一项长期艰巨的任务，有效的方法为肝移植，因肝脏供体少，临床工作难以大量展开，越来越多的研究者致力于研究能替代肝移植的方法，
2、间充质干细胞成为新的方向。 间充质干细胞(Mesenchymal stem cells，MSCs)属于干细胞家族，主要存在于骨髓、脐带、脐血和胚胎中。因具有多向分化潜能、造血支持、免疫调控和自我复制等特点，受到人们的关注。间充质干细胞在体内或体外诱导条件下可以分化为骨、软骨、脂肪、肌肉、肌腱、神经、类肝细胞、心肌等组织细胞，连续传代后仍具有多向分化潜能，可作为理想种子细胞用于炎症病变及衰老引起的器官修复。骨髓间充质干细胞应用于临床较早，但抽取骨髓为有创性检查，增加了病人痛苦，越来越多的学者致力于研究脐带来源的间充质干细胞。 脐带间充质干细胞来源广泛， 收集脐带为无创操作，且治疗肝硬化有促进肝脏
3、再生、提高患者生活质量的作用，在临床上应用越来越受到重视。 目的：制备足月顺产胎儿脐带来源的间充质干细胞，探讨体外分离、培养、鉴定方法，观察脐带间充质干细胞治疗终末期肝硬化的临床疗效。 方法：无菌条件下采集新生儿脐带，经过分离，培养获得间充质干细胞，流式细胞仪表面抗原测定（CD29、CD105、CD34、CD45）；选择50例肝硬化患者，26例患者在内科治疗基础上经前臂静脉或肝动脉插管进行MSCs移植，术后定期检测肝功能、凝血功能及临床症状，并观察同期的症状体征和不良反应，同时随机选择24例仅以内科治疗的肝硬化患者做为对照组，两组间进行比较。 结果： III(1) 培养的脐带间充质干细胞呈长梭
4、形、旋涡状生长。P1代消化后传代，贴壁时间约48h左右，2-3W长满80。P6代以内的细胞增殖无明显差异。P3-P6代生长速度较快，24h贴壁，48h长满70，72h长满80,P3-P6代内无明显分化。 (2) 细胞免疫表型显示表达间充质干细胞的特异性标记CD29；但不表达造血干细胞表面标记CD34、CD45。这与骨髓间充质干细胞的表型一致。 (3) 脐带间充质干细胞移植后，治疗组 ALB、PA 和 Fbg 水平比对照组明显升高(P0.05),PT 时间与对照组相比明显降低 (P0.05)。 (4) 治疗组患者从治疗后1周开始，乏力、腹胀、纳差明显好转，12周后患者腹水明显减少。26例移植患者
5、术中未出现严重不良反应，无与移植相关的严重并发症。 结论： (1) 本实验用双酶消化法分离出间充质干细胞，经过冻存、复苏后细胞损伤小，多次传代后细胞分化少，增殖能力稳定。 (2) 人脐带间充质干细胞经过流式细胞仪测定后，表达间充质干细胞的表型，不表达造血干细胞表型，与骨髓来源的间充质干细胞检测一致。 (3) 人脐带间充质干细胞治疗终末期肝硬化，可提高患者ALB、PA、Fbg，缩短PT，改善了患者的临床症状。 (4) 人脐带间充质干细胞移植肝硬化患者，具有良好的安全性。 关键词：脐带,间充质干细胞,移植,肝硬化 VABSTRACT Hepatic cirrhosis are caused by
6、a variety of liver damage, liver cells showing degeneration and necrosis, fibrosis, nodular regeneration, lobular architecture distortion, blood circulation route to be converted, eventually leading to cirrhosis. Based on etiology, hepatic cirrhosis are classified to viral cirrhosis, alcoholic cirrh
7、osis, parasitic liver cirrhosis, toxic cirrhosis, biliary cirrhosis, autoimmune cirrhosis, congestive cirrhosis, cryptogenic cirrhosis and so on. Hepatic cirrhosis caused by Hepatitis B virus is very common in China. According to statistics, about 9,300 million people carry hepatitis B virus, of whi
8、ch about 30 million are hepatitis B patients. Due to lack of effective treatment for hepatitis B virus and high cost of treatment, many patients can not receive long-term treatment ultimately, and eventually lead to cirrhosis. Patients of hepatic cirrhosis often suffer from portal hypertension and l
9、iver dysfunction, and finally result in death due to hepatic encephalopathy and gastrointestinal bleeding. Therefore, the treatment of hepatic cirrhosis is a difficult task. Effective therapy for hepatic cirrhosis is liver transplantation. But it is hard to be carried out in clinical work for lack o
10、f liver donor. More and more scientists focus on some other methods replacing liver transplantation. Mesenchymal stem cells is one of the options for treatment of hepatic cirrohsis. Mesenchymal stem cell is a member of stem cells. Mesenchymal stem cells are mainly in the bone marrow, umbilical cord,
11、 cord blood, embryos. Mesenchymal stem cells received much attention because of multiple differentiation potential, hematopoietic support, immune regulation, self-replication and other characteristics. Mesenchymal stem cells can serve as ideal seed cells for inflammation caused by disease and organ
12、repair due to aging. In vivo or in vitro conditions, MSCs can be induced and differentiated into bone, cartilage, fat, muscle, tendon, nerve, liver cells, heart and so on. MSCs continuous passage has multiple differentiation potential. Mesenchymal stem cells are very early to be used clinically. Bon
13、e marrow extraction is invasive and increase patients，suffering. A growing number of scholars have been working on the umbilical cord derived mesenchymal stem cells. Umbilical cords are easy to be collected VInon-invasively, and therefore umbilical mesenchymal stem cells are commonly used in treatme
14、nt of a variety of diseases in clinical practice, including liver cirrhosis. Objective: Cultivate mesenchyma stem cells the full-term childbirth embryo umbilical cord， explore the method of umbilical cord mesenchymal stem cells separation in vitro, proliferation, identification, and observe the effi
15、cacy in treating of end-stage hepatic cirrhosis. Methods: Collect newborn umbilical cord under sterile conditions, culture mesenchymal stem cells after separation, take cells for flow cytometry checking CD29, CD105, CD34, CD45. Select 50 cases of liver cirrhosis, 26 patients received basic medicatio
16、n and MSCs transplantation as well by way of forearm vein or the hepatic artery. Liver function, blood coagulation function, clinical symptoms, signs, symptoms and adverse reactions were monitored postoperatively. In addition, 24 patients with cirrhosis are randomly selected to receive regular medical treatment as control. Results: (1) Mesenchymal stem cells from umbilical cord are spiral-shaped growth in culture. Cells of passage 1 were digested and passaged. Its adherence time is about
