PTEN基因诱导人脑胶质瘤SHG-44细胞凋亡及bcl-2蛋白表达下调

目的　探讨PTEN基因对人脑胶质瘤SHG 44细胞凋亡及凋亡相关基因bcl 2表达的影响 ,阐明PTEN基因抑制肿瘤细胞增殖的机理。方法　PTEN基因体外转染SHG 44细胞 ,筛选阳性细胞克隆 ,以原位杂交、免疫组化方法检测PTEN基因的表达情况采用透射电镜、流式细胞仪和核DNA琼脂糖凝胶电泳检测细胞的凋亡情况以免疫荧光法检测bcl 2基因的表达。结果　PTEN基因转染的SHG 44细胞有PTEN基因和蛋白的表达。透射电镜下可见转染PTEN基因后细胞核染色质浓缩边集、胞浆浓缩、核碎裂及凋亡小体形成等典型的凋亡表现 ;流式细胞仪示细胞周期从G1 期到S期发生抑制 ,并且在G1 期峰前出现一明显的凋亡峰 (1 2 .9% ) ;细胞核DNA琼脂糖凝胶电泳呈现凋亡细胞特有的梯状条带 ;bcl 2的表达也显著下调。转染空载体和未转染的SHG 44细胞无明显的凋亡表现。结论　PTEN基因可诱导SHG 44细胞凋亡 ,并下调bcl 2蛋白的表达 ,这可能是PTEN基因抑制肿瘤细胞增殖的分子机制之一

PTEN gene induces apoptosis and Down-regulation of bcl-2 protein in human brain glioma SHG-44 cells

Objective To investigate the effects of PTEN gene on the apoptosis and expression of gene related with apoptosis in human brain glioma SHG 44 cells ,and to probe for the mechanism that PTEN gene inhibits the proliferation of tumor cells.Methods SHG 44 cells were transfected in vitro with a plasmid vector containing PTEN gene .and then positive cell clones were selected and amplified .Expression of PTEN gene was examined by in situ hybridization and immunohistochemistry methods.Cells were examined by transmission electronic microscope,flow cytometry and nucleus DNA agarose gel electrophoresis to detect the apoptosis in them.The expression of bcl 2 gene was detemined by innuno fluorescence method.Results SHG 44 cells transfected with PTEN gene were obtained by selection,and expressed the PTEN gene and protein.Changes of ultrastructure included peripheral accumulation of nuclear chromatin,fragmentation of nucleus and apoptosis body.Cell cycle determined by flow cytometry showed that the percentage of G 1 phase increased,but the percentage of S phase decreased.One visible apoptosis peak(12.9%)in front of the G 1 peak was found by FCM.The characteristic DNA ladder was found in the agarose gel electrophoresis of the nucleus DNA of tranfsected cells.Expression of bcl 2was evidently down regulated in these cells.The untransfected cells and cells transfected with empty vector had no abnormal per formance.Conclusion The transduction of PTEN gene into SHG 44 cells could induce apoptosis and down regulate bcl 2 in these cells,and may result in the inhibition on the proliferation of human tumor cells.