骨髓间充质干细胞多向分化潜能和微环境关系的研究

摘要

目的:深入研究骨髓间充质干细胞(mesenchymal stem cells,MSCs)多向分化的潜能与微环境的关系,从而为促进MSCs向目标组织细胞诱导分化创建新的实验方法.方法: 大鼠MSCs进行分离,体外培养、扩增、鉴定.将MSCs向脂肪细胞和胰岛细胞方向诱导分化,并深入研究在不同的微环境下,其分化能力的差别,对照组诱导剂为含有角朊细胞生长因子(KGF)、胰岛素-转铁蛋白-硒(ITS)、尼克酰胺的无血清DMEM/F12培养基,实验组在对照组基础上添加胰腺条件培养液;对诱导的胰岛细胞进行观察、双硫腙染色,并进行葡萄糖刺激实验,测定细胞分泌胰岛素及C-肽功能.结果: 培养的MSCs表现为非造血干细胞特性.其可向脂肪细胞,胰岛细胞等不同组织细胞分化.对照组和实验组均可分化为胰岛细胞,但实验组分化而成的胰岛细胞在数量和功能上均高于对照组.结论:MSCs具有多向分化潜能,其分化能力在特定的微环境下更强.%Objective :To investigate the relation of multipotentiality of hone marrow mesenchymal stem cells and microenvironment, To find a new method of promote inducing differentiation bone marrow mesenchymal stem cells ( MSCs)into objective cells in vitro.Methods: MSCs were isolated from SD rat, cultured in vitro, purified and then identified by testing the phenotypes with flow cytometry ( FCM) .MSCs was induced and differentiated into adipocyte and islet cells.To study the difference of MSCs differentiation ability under different microenvironments in vitro, control group was cultivated in serum free DMEM/F12 medium including keratinocyte growth factor( KGF) ,ITS and Nicoiinamide, while experiment group was cultivated in serum free DMEM/F12 medium in which the rat pancreatic extract was added.During the cultivation period the cells were taken for light microscopy at different time points, identified by dithizon( DTZ) .The glucose stimulation test was performed in vitro and the levels of insulin and C - peptide in response to the different glucose concentrations were assayed.Results : According to cell phenotypes with flow cytometry ( FCM) ,MSCs were non - hemopoietic stem cells.MSCs could differentiate into adipocyte, and MSCs could differentiate into islet cells both in the control group and expenmental group.The harvested cells from experiment group had greater number and better function than that from control group.Conclusion : MSCs had multipotentiality and the differentiation ability enhanced obviously under the specific microenvironment.