bcl-2及其重要功能结构域在调节成纤维细胞NIH3T3凋亡中的研究

目的 以成纤维细胞NIH3tT13作为细胞模型,探讨bel-2 Loop Domain结构域在成纤维细胞凋亡中的作用地位.方法 构建正常和Loop Domain结构域突变bcl-2基因的真核表达质粒,用于成纤维细胞转染;脂质体介导,分别将(1)空载体质粒(2)携带bcl-2基因质粒和携带突变基因的质粒转染成纤维细胞(NIH3T3),转染后用肿瘤坏死因子(TNF)-α刺激NIH3T3细胞诱导凋亡,应用流式细胞仪(FCM)检测各组细胞凋亡,应用华联小鼠全基因表达谱芯片检测各组细胞基因表达谱的变化.结果 成功构建bcl-2基因和Loop domain中突变的真核表达载体;成功将携带突变基因、野生基因的质粒转染入NIH3T3细胞;各组转染后用TNF-α刺激细胞诱导凋亡,FCM检测发现,TNF-α刺激细胞17 h后,NIH3T3未转染细胞凋亡明显增加,细胞凋亡率为(53.18 ±8.18)%,NIH3T3转染野生型bcl-2质粒组与NIH3T3未转染细胞组比较细胞凋亡率明显下调;NIH3T3转染突变bcl-2质粒组与NIH3T3未转染细胞组比较细胞凋亡率差异无统计学意义(P>0.05);NIH3T3转染野生型bcl-2质粒组与NIH3T3转染突变bcl-2质粒组比较细胞凋亡率明显上调;基因芯片检测发现:转染bcl-2基因的NIH3T3细胞凋亡基因与转染空质粒组下调,转染Loopdomain突变的真核表达载体的NIH3T3细胞凋亡基因比转染bcl-2基因的NIH3T3细胞上调.结论 证实bcl-2基因Loop domain结构域突变对bcl-2基因功能产生明显影响,bcl-2基因Loop domain结构域突变使NIH3T3细胞凋亡产生明显变化.

Detection of bcl-2 and its important function domain modulating fibroblasts apoptosis

Objective By using fibroblast line NIH3T3 as a cell model,role of bcl-2 loop domain in fibroblast apoptosis was discussed. Methods Normal and loop domain mutation bcl-2 eukaryotic expression plasmids were constructed,and transfected into fibroblasts. through liposome empty vector plasma, taking bcl-2 plasma and bcl-2 mutation plasma were transfected to fibroblast cell (NIH3T3). Then NIH3T3 cell was induced apoptosis by TNF-astimulation,and cell apoptosis was examined by flow cytometer. Cell gene expression chart' s change were detected by Hua Lian mouse gene chip. Results bcl-2 and bcl-2 loop domain mutation eukaryon expression plasma was successively constructed. Successively transfected bcl-2 wild and mutation type plasma to NIH3T3 cell,cell were stimulated and induced apoptosis by TNF-α. Detected by flow cytometer,after TNF-α stimualting cell inducing apoptosis 17 hour,NIH3T3 no transfected cell apoptosis upregulated markedly,cell apoptosis rate (53. 18 ±8. 18)% ,and apoptosis rate of transfecting wild type bcl-2 was downregulated than NIH3T3 no transfected cell. Apoptosis rate of transfecting mutation bcl-2 plasma had no markedly difference than NIH3T3 no transfected cell. Cell apoptosis rate of transfecting wild bcl-2 plasma was upregulated than NIH3T3 transfected mutation bcl-2 plasma. Gene chip detection result:cell apoptosis gene transfected bcl-2 gene was downregulated than transfected empty plasma. Cell apoptosis gene transfected mutation bcl-2 gene was upregulated than transfected bcl-2 gene. Conclusion bcl-2 loop domain mutation had markedly effection on bcl-2 gene function,and bcl-2 loop domain mutation made changes on NIH3T3 cell apoptosis.