骨髓间充质干细胞性状稳定性与生物安全性研究

Study on stability of biological characteristics and biosafety of BMSCs

目的:探讨长期体外培养的大鼠骨髓间充质干细胞(BMSCs)的生物学性状稳定性与应用安全性。方法:采集大鼠骨髓细胞用原代贴壁培养法获取大鼠BMSCs,进行长期体外传代培养后行流式细胞术检测第4、20和40代细胞周期变化和特异表面标志抗原表达改变;利用血清依赖实验和软琼脂克隆形成实验检测不同培养时期BMSCs成瘤潜能;以鼠源性C6胶质瘤细胞系为阳性对照,SD大鼠颅内种植等细胞数量BMSCs,MRI与鼠脑病理切片检测BMSCs体内成瘤性。结果:采用原代贴壁培养法获取稳定传代大鼠BMSCs,流式细胞周期分析显示BMSCs随传代次数增加细胞增殖速度有所增快,但流式细胞术表面标志抗原表达无明显改变;血清依赖实验和软琼脂克隆形成实验显示BMSCs较C6胶质瘤细胞系表现出极差的低营养耐受能力和半固体介质克隆形成能力;MRI与鼠脑病理切片未检测到BMSCs在体内的肿瘤形成。结论:长期体外培养的大鼠BMSCs生物学性状稳定,无致瘤潜能,体内移植相对安全。

Objective：To investigate the stability of biological characteristics and biosafety of BMSCs after a long period culture.Methods：Rat BMSCs derived from bone marrow cells were isolated and expanded in monolayer culture.Specific surface antigens and cell cycle kinetics of the 4th,20th and 40th passage of BMSCs were detected with flow cytometry.Serum-dependent assay and soft agar colony assay were used to examine the tumorigenesis of the long term cultured BMSCs in vitro.Then employing the rat-derived C6 glioma cell line as positive control,the same number of BMSCs were implanted into SD rat brain,MRI and histopathological examination of rat brain were used to evaluate the tumorigenicity of BMSCs in vivo.Results：Stably passaged rat BMSCs were obtained by monolayer culture of bone marrow cells as identified by their specific surface antigens.The cell growth rate of BMSCs was increased slightly with the increasing passages of cell culture.However,the expression of specific surface antigens and DNA ploidy did not alter significantly through flow cytometric analysis.The results from serum-dependent assay and soft agar colony assay demonstrated that the proliferation of BMSCs was highly serum-dependent and they had poor ability of colony formation in semisolid medium as compared with C6 glioma cells,MRI and histopathological examination of rat brain also showed that BMSCs did not form tumors in vivo.Conclusion：Rat BMSCs in a long period culture have stable biological characteristics and do not exhibit tumorigenic potency both in vitro and in vivo.It should be safe to apply for transplantation.