体外诱导人骨髓间充质干细胞向肝系细胞定向分化实验研究

发布时间：2018-01-03 05:26

  本文关键词：体外诱导人骨髓间充质干细胞向肝系细胞定向分化实验研究　出处：《南昌大学》2006年硕士论文　论文类型：学位论文

  更多相关文章： 骨髓间充质干细胞 胎肝细胞 肝细胞生长因子 肝细胞 分化

【摘要】：目的：骨髓间充质干细胞(Mesenchymal stem cells，MSCs)是一种存在于骨髓内的非造血干细胞，由于其具有自我更新能力及多向分化潜能，在组织器官修复及再生方面显示出良好的应用前景而倍受研究者关注。本实验通过体外培养MSCs，了解其生长生物学特性；探讨其向肝系细胞分化的可能性，观察肝细胞生长因子(hepatocyte growth factor HGF)，碱性成纤维细胞生长因子(basic fibroblast factor bFGF)及人胎肝细胞对培养的MSCs的诱导作用，寻找影响和调节MSCs向肝细胞分化的因素，探讨体外诱导培养获取肝细胞的可行性。以期为临床肝细胞移植及生物人工肝治疗提供新的细胞来源。 方法： 1．采用密度梯度离心法分离MSCs，反复贴壁法纯化扩增MSCs，镜下观察其形态学特点。 2．采用胶原酶Ⅰ型消化法分离人2-4月胚胎肝脏细胞，差速贴壁法纯化肝脏细胞后培养。 3．通过加入一定浓度生长因子HGF和bFGF对MSCs诱导培养，未加入生长因子组作为对照组。观察诱导细胞的形态学变化。采用免疫细胞化学染色技术检测诱导培养细胞的甲胎蛋白(alpha-fetoprotein AFP)、细胞角蛋白-18(cytokeratin-18 CK-18)、细胞角蛋白-19(cytokeratin-19 CK-19)等肝系细胞标志物的表达情况及进行糖原染色观察诱导细胞的糖原合成及贮存功能，同时留取不同时段的细胞培养上清液用放射免疫法检测AFP浓度以观察诱导细胞的AFP合成及分泌功能。 4．MSCs标记Hoechst33342后，将MSCs和人胎肝细胞直接混合培养和在双室培养皿中半透膜隔开混合培养。胎肝细胞培养组及MSCs培养组作为对照组。观察诱导细胞的形态学变化，采用免疫细胞化学染色技术检测诱导培养
[Abstract]:Objective: bone marrow mesenchymal stem cells (MSCs) is a non-hematopoietic stem cell in bone marrow. Because of its self-renewal ability and multi-differentiation potential, it has shown a good application prospect in tissue and organ repair and regeneration. In this experiment, MSCs was cultured in vitro. To understand the biological characteristics of its growth; To investigate the possibility of hepatocyte growth factor differentiation. Induction of MSCs by basic fibroblast factor bFGF and human fetal hepatocytes. To find out the factors that influence and regulate the differentiation of MSCs into hepatocytes, and to explore the feasibility of inducing and culturing hepatocytes in vitro in order to provide a new cell source for clinical hepatocyte transplantation and bioartificial liver therapy. Methods: 1. MSCs were separated by density gradient centrifugation, then purified and amplified by repeated adherent method. The morphological characteristics of MSCs were observed under microscope. 2. The hepatocytes of 2-4 months embryo were isolated by collagenase 鈪，

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