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

Abstract

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. Key words: Fibroblast cell; bcl-2; Loop domain; Apoptosis