骨髓间充质干细胞定向分化肝细胞并肝内移植及软肝煎应用对肝纤维化形成大鼠肝功能影响的研究

发布时间：2019-01-12 17:26

【摘要】：再生医学是本世纪在生物医学史上重要工作之一。肝脏再生及干细胞的作用一直是大家研究的一个热点。目前干细胞移植研究多集中在胚胎干细胞、骨髓干细胞等,由于胚胎干细胞存在着伦理、法律、免疫排斥反应等问题,难以用于临床。自体骨髓来源肝细胞移植作为新的治疗肝衰竭病人方法已被人们所关注。最近研究表明,骨髓间充质干细胞(Mesenchymal stem cells,MSCs)具有多向分化潜能,包括向神经、血液、心肌、肝细胞等分化,这些引起了专家们对骨髓间充质干细胞可塑性的极大兴趣。骨髓来源肝细胞移植不仅解决自体肝细胞获取困难、增殖性差等问题,同时为肝脏再生组织工程研究提供了一个平台。为了探索骨髓间充质干细胞横向分化为肝细胞的作用机制,研究骨髓源性的肝细胞肝内移植定向分化,我们向肝纤维化形成大鼠肝脏内移植经诱导的骨髓间充质干细胞来源的肝细胞,观察其分布、分化情况,并观察联合应用中药软肝煎对肝纤维化形成大鼠肝脏功能改善的效果。一.首先我们从SD 大鼠的股骨中提取骨髓干细胞,利用Percoll 分离液密度梯度离心法结合贴壁法实现骨髓间充质干细胞的体外分离纯化、培养、传代扩增。取第3 代的骨髓间充质干细胞,应用流式细胞仪检测细胞表面抗原CD44、CD34、CD45、CD31、CD90,对骨髓间充质干细胞进行鉴定。流式细胞仪观查结果显示CD34、CD45、CD31 阴性,CD44、CD90 阳性。表明提取、分离、培养是非骨髓造血细胞的骨髓间充质干细胞。取第3 代的骨髓间充质干细胞以1×106/cm2的密度分别种植在六孔培养板中,并加入肝细胞生长因子(HGF)终浓度为20ng/ml 表皮生长因子(EGF)终浓度为1.5μg /ml,进行定向肝细胞诱导分化,诱导过程中用倒置相差显微镜观察细胞形态变化。对诱导产生的细胞,应用免疫荧光法进行肝细胞特异性标志CK8、CK18 和白蛋白检测。其中细胞检测出有CK8、CK18 和
[Abstract]:Regenerative medicine is one of the important works in the history of biomedical science in this century. The role of liver regeneration and stem cells has been a hot topic. At present, stem cell transplantation is mainly focused on embryonic stem cells, bone marrow stem cells and so on. Because of the ethics, law, immune rejection and other problems, embryonic stem cells are difficult to be used in clinical practice. Autologous bone marrow derived hepatocyte transplantation as a new treatment for patients with liver failure has been paid attention to. Recent studies have shown that bone marrow mesenchymal stem cells (Mesenchymal stem cells,MSCs) have the potential to differentiate into nerve, blood, myocardium and hepatocytes, which has aroused great interest in the plasticity of bone marrow mesenchymal stem cells. Bone marrow-derived hepatocyte transplantation not only solves the difficulties of obtaining autologous liver cells, but also provides a platform for the study of liver regeneration tissue engineering. In order to explore the mechanism of transversely differentiation of bone marrow mesenchymal stem cells into hepatocytes, and to study the differentiation of bone marrow-derived hepatocytes in liver transplantation. Liver cells derived from induced bone marrow mesenchymal stem cells were transplanted into the liver of rats with hepatic fibrosis. The distribution and differentiation of liver cells were observed. The effect of the combination of Chinese medicine Ruangan decoction on the liver function of rats with hepatic fibrosis was observed. I. Firstly, bone marrow stem cells were extracted from the femur of SD rats. Bone marrow mesenchymal stem cells were isolated, cultured and amplified by Percoll density gradient centrifugation combined with adherent method in vitro. Bone marrow mesenchymal stem cells (BMSCs) of the third passage were identified by flow cytometry (FCM) detection of cell surface antigen (CD44,CD34,CD45,CD31,CD90,). Flow cytometry showed that CD34,CD45,CD31 was negative and CD44,CD90 was positive. These results indicate that bone marrow mesenchymal stem cells (BMSCs) that are not bone marrow hematopoietic cells are extracted, isolated and cultured. Bone marrow mesenchymal stem cells (BMSCs) of the third passage were planted in a six-well culture plate with a density of 1 脳 106/cm2, and the final concentration of hepatocyte growth factor (HGF) was 1.5 渭 g / ml, when the final concentration of 20ng/ml epidermal growth factor (EGF) was 1.5 渭 g / ml,. The differentiation of hepatocytes was induced and the morphologic changes of the cells were observed by inverted phase contrast microscope. The hepatocyte specific markers CK8,CK18 and albumin were detected by immunofluorescence. Among them, the cells detected the presence of CK8,CK18 and
【学位授予单位】：大连医科大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R329;R285

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