【作者】 唐力军；

【导师】 高毅；

【作者基本信息】 第一军医大学 ， 肝胆外科， 2004， 硕士

【摘要】 肝炎、肝硬化及肝癌等肝脏疾病所至的终末期肝功能障碍，目前最有效的治疗手段是原位肝移植(OLT)。然而，由于供体的缺乏、免疫排斥和高额费用等问题，OLT的广泛应用受到了限制。杂交型生物人工肝(HBAL)的目的是使终末期肝病病人桥接(bridge)肝移植，而且肝细胞具有强大的再生能力，临时应用生物人工肝也可以解决急性肝功能的失代偿，促进肝细胞再生，部分患者肝功能完全自然恢复，病情康复。但由于作为HBAL核心生物材料的肝细胞来源问题未得到根本解决，使HBAL的临床应用远未达到理论上的预期效果。研究证明人骨髓来源的多能成体祖细胞(MAPCs)具有向肝样细胞分化的潜能，由于其具有易于调控、供源丰富、容易获取、有自体供源、避免免疫排斥等优点，因此，有望成为理想的肝组织工程细胞来源。 第一部分：人骨髓来源的多能成体祖细胞分离纯化及体外培养的实验研究 目的 建立体外分选纯化人MAPCs的方法，用自行配制的细胞培养基对MAPCs进行体外培养，了解其生长、增殖特性。 方法 取志愿者适量骨髓后采用梯度密度离心法分离获取单个核细胞，对单个核细胞行贴壁培养后，将获取的骨髓贴壁细胞通过CD45、GlyA免疫微磁珠负分选，流式细胞仪鉴定分选后细胞纯度；培养观察细胞生长情况，对传代到不同阶段的细胞进行CD45、GlyA流式细胞分析，初步了解MAPCs在培养、增殖过程中的免疫学稳定性。 结果 (1)通过MACS分选，平均每1×10~6／ml骨髓贴壁细胞可分选出约5—10×10~4／ml数量级的MAPCs，分选前后细胞活力分别为(96.7±1.7)％和(96.0±2.4)％，无明显差异；(2)用我们自制的培养基培养分选后的MAPcs，细胞生长良好，最长传代到第16代;(3)流式细胞仪分析获取的CD45一、GlyA一细胞纯度大于98%;流式细胞仪分析传代第4、8、12代MA卫Cs，eD45一、GlyA一细胞纯度大于95%。 结论(1) CD45、GlyA免疫微磁珠负分选可从骨髓中分离高纯度的M妙cs;(2)MAPcs在体外有较强的增殖能力，且能保持较长时间的稳定状态;(3)我们自行研制的人MAPCs培养基能成功体外培养MAPCS。第二部分:人骨髓来源的多能成体祖细胞在不同诱导条件下向肝样细胞 分化的实验研究 目的探索人骨髓来源MAPCs在不同条件下诱导分化为肝细胞的可行性;摸索并确立骨髓MAPCs诱导为肝细胞的适当培养方法及条件，建立一个获得“骨髓源MAPCs”的成熟技术平台，为其在组织工程学研究和临床医学中的广泛应用奠定基础。 方法(一)分组:A组:H邵(Zong/ml)、B组:FGF一4(10ng/ml)、C组:HGF(Zong/ml)+FGF一4(long/ml)、D组:24小时70%肝叶切除兔门静脉血清(RPVS，10%浓度)诱导分化MAPCs，E组(阳性对照组):L一02人肝细胞株、F组(阴性对照组):未加任何诱导因素的MAPCs; (2)免疫细胞化学、免疫荧光化学鉴定各组不同诱导分化阶段细胞的ALB、AFP、CK一18、CK一19等肝细胞特征的表型变化并计数阳性细胞比率;(3)RT一 CR检测A、B、C组不同诱导分化阶段细胞的ALB、AFP、e玲18、eK一19的mRNA转录;(4)westem blot检测B、e组诱导分化第21天、35天后细胞的ALB的表达。 结果(l)免疫细胞化学、免疫荧光化学结果:ALB、CK-18在A、B、c、D、E组中基本为阳性着色;cK一19均为阴性着色，仅D组有阳性着色;AFP作为早期不成熟肝细胞的一个标志，在A、B、C组的诱导早期基本为阳性着色，在诱导中、晚期基本为阴性着色，仅D组在诱导中、晚期有阳性着色。计数阳性细胞比率显示:HGF及FGF一4均能诱导MAPcs向肝样细胞分化，HGF+FGF一4诱导强于二种细胞因子单独诱导，我们自行制作的即VS诱导分化能力最强，各种肝细胞表型标志表达的阳性率最高;(2)RT一CR结果:作为不成熟肝细胞表型的AFP，在诱导分化的第7天，A、B、C组mRNA均有阳性表达，CK-19在分化的第21天mRNA均为阴性;作为成熟肝细胞表型的ALB及CK一18，各组在不同时间段mRNA的表达基本都为阳性;(3) Westemblot检测结果:B、C组在诱导分化第21天、35(38)天，ALB均有阳性表达。 结论(l)人骨髓来源的MAPCs具有向肝样细胞分化的潜能;(2)HGF及FGF一4具有诱导人骨髓来源的MAPCs向肝样细胞分化的能力，HGF+FGF一4诱导强于二种细胞因子单独诱导;(3)24小时70%肝叶切除兔门静脉血清(对Vs)具有诱导人骨髓来源的MAPCs向肝样细胞分化的能力，且强于细胞因子诱导。更多还原

【Abstract】 The treatment of liver diseases, such as hepatitis, liver cirrhosis and liver cancer, is always a global difficulty. To end-stage liver failture led by these disorders, orthotopic liver transplantation(OLT) is the only established treatment, but it is associated with donor shortage, immune rejection, and high cost, which are the limitations of this therapy. The treatment purpose of Hybrid bioartificial liver(HBAL) to the end-stage liver failure patient is bridged for OLT. On the other hand, the function of hepatocyte repopulation is mighty, applied HBAL temporarily may obtain some purpose of treating anxious liver function, accelerating hepatocyte repopulation, supporting liver function renenw, and healing the state of an illness. However, the anticipated clinical efficacy of HBAL has not proved yet, mainly because of the source deficiency of hepatocytes which are the core bioartificial materials of HBAL.The investigation have proved that multipotent adult progenitor cells(MAPCs) from human bone marrow can differentiate into functional hepatocyte-like cells. MAPCs may be a relative ideal cells for liver tissue engineering as they are more easily to be manipulated, have the merits of abundant donor source and easy to harvest, and be obtainted autologously to avoid immune rejection. Part I : The investigations on purification and culture of multipotentadult progenitor cells from human bone marrow Objective To create a method of separating human MAPCs in vitro using immunomagnetic beads. To investigate the possibilities of MAPCs-5-producting and proliferating by cell culture produced by ourselves.Methods The bone marrow mononuclear cells were obtained by inspiring the proper volume of bone marrow from volunteers and then centrifuging by gradient and density. The mononuclear cells were cultivated by adherent culture. Collected the bone marrow adherent cells, and isolated the adherent cells by MACS through depletion selection by use of CD45 and GlyA microbeads. Analyzing the component of the CD45~>. GlyA" cells by flow cytometers. Observed the cells’ form hi different growing phase. Flow cytometers analyzed the subculture cells at different time point in order to investigate the immunization stability of MAPCs producting and proliferating by cell culture.Results (1) Per lX106/ml bone marrow mononuclear cells could separated 5 ?10X104/ml MAPCs by MACS. The activity of the pro-purification or after purification cells was (96.7?.7) %and (96.0?.4) % respectively, which was not different significantly with that before purification. (2) The MAPCs grew well in the cell cultures produced by ourselves. We observed that MAPCs could be expanded for more than 16 population doubling. (3) The purity of the CD45~% GlyA~cells separated from bone marrow adherent cells was more than 98 % by flow cytometers. The purity of MAPCs which had cultivated 4, 8 and 12 cell doublings were more than 98 % by flow cytometers.Conclusions (1) The MAPCs derived from bone marrow can be purified by MACS through depletion selection by use of CD45 and GlyA microbeads, and they can survive and differentiate after tune. (2) The abilities of MAPCs producting and proliferating by cell cultures is active, and can keep non-differentiation state for a long time. (3) MAPCs cultures produced by ourselves is appropriate MAPCs culture in vitro.Part II: The investigations of multipotent adult progenitor cells fromhuman bone marrow differentiating into hepatocyte-like cells in thedifferent appropriate condition in vitroObjective To investigate the possibility of the MAPCs from human-6-bone marrow which are induced into hepatocytes in vitro. To explore the feasibility of differentiation from the MAPCs into the hepatocytes, and to establish the method and condition of MAPCs culture in vitro. And then, to make the hepatocytes derived from MAPCs as germ cell to be useable in tissue-engineering and clinical medical.Methods (l)Divided groupsrA: Hepatocyte growth factor (HGF, 20ng/ml), B: Fibroblast growth factor-4(FGF-4 , 20ng/ml),D: HGF(20n更多还原