Bcl-2基因转染对骨髓间充质干细胞体外模拟缺血性损伤保护作用的实验研究  

The protective effect of Bcl-2 gene transfection on mesenchymal stem cell injury caused by analogous ischemia in vitro

作　　者：张小乔[1] 梅元武[1] 张红[1] 郑丽芳[1] 郭远瑾[1] 徐江华[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院神经内科

出　　处：《卒中与神经疾病》2008年第3期170-174,共5页Stroke and Nervous Diseases

摘　　要：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 survey the protective effect of Bcl-2 gene transfection on mesenchymal stem cells （MSCs） injury caused by analogous ischemia in vitro. Methods Density gradient centrifugalization combined with adherence method were used to segregate and cultivate rat MSCs. MSCs cultivated to the 5th passage were characterized using immunocytochemistry technology. Liposome tranfection method was used to transfect MSCs with human Bcl-2（hBcl-2） gene and the transfected MSCs were sieved with G418. Then immunofluorescence cytochemistry method was used to detect the hBcl-2 protein expression of gene modified MSCs. Serum and oxygen deprivation was used to cause analogous ischemia injury of MSCs in vitro. Flow cytometry and TUNEL in situ apoptosis detection method were used to evaluate apoptosis and survival of MSCs and hBcl- 2 gene transfected MSCs. Results Density gradient centrifugalization combined with adherence method could segregate and purify rat MSCs effectively. The results of immunocytochemistry showed that CD44 and CD71 expression were positive while CD45 expression was negative for MSCs. MSCs transfected with hBcl-2 gene and sieved with G418 showed stable and high effective expression of hBcl-2 protein. The results of flow cytometry and TUNEL showed that the number of apoptosis cell of Bcl-2 transfected MSCs was less and the number of survival cell was more than that of nontransfected MSCs after 6h,9h serum and oxygen deprivation. Conclusions MSCs transfected with hBcl-2 gene presented stable hBcl-2 protein expression, hBcl-2 gene transfection can antagonize MSCs apoptosis caused by analogous ischemia in vitro thus had protective effect.

关 键 词：Eel-2 基因转染 骨髓间充质干细胞 缺血性损伤 保护作用 

分 类 号：Q813[生物学—生物工程] R743[医药卫生—神经病学与精神病学]