骨髓间充质干细胞体外培养及肝向分化方案的优化

发布时间：2018-03-11 16:13

本文选题：骨髓间充质干细胞　切入点：体外培养　出处：《南方医科大学学报》2015年08期 　论文类型：期刊论文

【摘要】：目的优化一套简便、可靠、稳定的大鼠骨髓间充质干细胞分离、培养、鉴定及肝向分化的方案。方法采用全骨髓差速贴壁法分离提取大鼠骨髓间充质干细胞,通过优化的1.5 h差速贴壁结合12 h首次全量换液方法,及体外改良培养方案实现细胞高效纯化扩增。流式细胞术表面标志物检测结合成脂、成骨、成软骨诱导分化鉴定细胞群。采用添加b FGF、HGF、EGF等多种生长因子的三步诱导法促使骨髓间充质干细胞向肝系分化,并进行形态学、免疫学、基因水平评估。结果分离纯化的细胞群阳性表达CD29、CD44、CD90,阴性表达CD34、CD45,经成脂、成骨、成软骨诱导液作用后,油红O、茜素红、甲苯胺蓝染色均呈阳性。经三步法肝向诱导处理后,细胞群呈肝样细胞形态改变,表达肝脏特异性标志物ALB、AFP,肝系相关基因表达水平呈时间依赖性逐渐上升。结论优化后的方案能够简便、可靠、稳定地获得高纯度的大鼠骨髓间充质干细胞,并能在特定微环境作用下实现肝向分化。
[Abstract]:Objective to optimize a set of simple, reliable, stable rat bone marrow mesenchymal stem cell isolation, culture, identification and differentiation of liver program. By using the whole bone marrow differentialadherence was isolated from rat bone marrow mesenchymal stem cells, through the optimization of 1.5 h differential adherent combined with 12 h for the first time around the amount of liquid change method, and in vitro modified culture scheme cells highly purified and amplified. Flow cytometry surface markers and adipogenic, osteogenic, chondrogenic differentiation of cells. By adding B FGF, HGF, EGF and other growth factor induced by three step method to promote bone marrow mesenchymal stem differentiation to hepatic cells, and the morphology, immunology and gene level evaluation. Results the purified cells were positive for CD29, CD44, CD90, CD45, CD34 negative expression, the adipogenic, osteogenic, chondrogenic media after oil red O, alizarin red, toluidine blue staining was positive. The three step to the liver after treatment, cells showed morphologic changes of hepatocyte like cells, expression of liver specific markers ALB, AFP, liver related gene expression in a time-dependent manner gradually increased. Conclusion the optimized scheme can be simple, reliable and stable to obtain high purity of rat bone marrow mesenchymal stem cells and, in a specific micro environment under the action of liver differentiation.

【作者单位】：西安交通大学第一附属医院肝胆外科;西安交通大学先进外科技术与工程研究所;陕西省人民医院肿瘤外科;
【基金】：教育部高等学校博士学科点专项科研基金(20110201130009) 陕西省科学技术研究发展计划国际合作项目(2012KW-39-01)
【分类号】：R575

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