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目的观察Bcl-2基因转染对骨髓间充质干细胞(MSCs)体外模拟缺血性损伤的保护作用,从而为下一步Bcl-2基因修饰的MSCs移植治疗脑缺血的实验研究奠定基础。方法采用密度梯度离心结合贴壁法分离、培养大鼠MSCs,对培养第5代的MSCs进行鉴定;用脂质体转染法对MSCs行人Bcl-2(hBcl-2)基因转染,转染后用G418进行筛选,然后用免疫荧光细胞化学技术检测其蛋白的表达;采用血清剥夺和缺氧(SOD)处理建立MSCs体外模拟缺血性损伤模型,通过流式细胞术和TUNEL原位细胞凋亡检测评价转染hBcl-2基因的MSCs和未转染的MSCs的凋亡及存活情况。结果密度梯度离心结合贴壁法能有效分离、纯化大鼠MSCs,免疫组化检测显示培养的细胞CD44、CD71表达阳性,而CD45表达阴性;转染hBcl-2基因的MSCs经G418筛选后有hBcl-2蛋白的稳定、高效的表达;流式细胞术检测显示和MSCs相比,转染hBcl-2基因的MSCs在SOD后及9h凋亡细胞较少,存活细胞较多。TUNEL原位细胞凋亡检测也显示转染hBcl-2基因的MSCs的凋亡明显减少(P<0.05)。结论转染hBcl-2基因的MSCs有其蛋白的稳定表达,hBcl-2基因转染可对抗MSCs体外模拟缺血性损伤引起的细胞凋亡,从而具有保护性作用。
Objective To observe the protective effect of Bcl-2 gene transfection on simulated ischemic injury of bone marrow mesenchymal stem cells (MSCs) in vitro, and lay the foundation for the experimental study of Bcl-2 gene modified MSCs transplantation in cerebral ischemia. Methods Rat MSCs were isolated and cultured by density gradient centrifugation and adherence method. MSCs of passage 5 were identified. Human hBcl-2 gene was transfected into human MSCs by lipofectamine. Then the cells were screened by G418 and then detected by immunofluorescence cytochemistry. MSCs were established by serum deprivation and hypoxia (SOD) in vitro and the model of ischemic injury was established by flow cytometry and TUNEL in situ Apoptosis and survival of MSCs transfected with hBcl-2 gene and non-transfected MSCs were evaluated by apoptosis assay. Results MSCs were isolated and purified by density gradient centrifugation and adherence method. Immunohistochemistry showed that CD44 and CD71 were positive in cultured cells but negative in CD45. MSCs transfected with hBcl-2 gene were screened by G418 for hBcl The results of flow cytometry showed that compared with MSCs, MSCs transfected with hBcl-2 gene had fewer apoptotic cells and more viable cells at 9h after SOD. TUNEL in situ cell apoptosis assay also showed that the apoptosis of MSCs transfected with hBcl-2 gene was significantly reduced (P <0.05). Conclusion MSCs transfected with hBcl-2 gene have stable expression of their proteins. HBcl-2 gene transfection can protect MSCs against apoptosis induced by ischemic injury in vitro and thus have a protective effect.