【摘要】 目的研究抑癌基因PTEN对人膀胱癌细胞株BIU-87失巢凋亡的影响并探讨其可能的机制。方法将携有野生型PTEN基因及2种突变型基因C124A-PTEN和G129E-PTEN的真核表达载体转染BIU-87细胞,利用West-ernblot法,检测PTEN蛋白表达与蛋白激酶B(PKB/Akt)、焦点黏附激酶(FAK)磷酸化水平的变化,并应用流式细胞仪技术和激光共聚焦显微镜技术,分析各种细胞在黏附与失黏附状态下的凋亡。结果与对照组相比,转染野生型PTEN的BIU-87细胞中,FAK和Akt的磷酸化水平分别降低了59%(P<0.01)和89%(P<0.01),且失巢凋亡率由(8.32±0.57)%增至(37.62±2.12)%;G129E-PT/87细胞内磷酸化FAK和磷酸化Akt的水平分别下降了62%(P<0.01)和46%(P<0.05)且失巢凋亡率为(32.57±1.72)%;而转染C124A-PTEN的BIU-87细胞中,FAK和Akt的磷酸化水平及失巢凋亡率均无明显变化。结论抑癌基因PTEN可依赖其磷酸酶活性抑制FAK和Akt的磷酸化,并诱导膀胱癌细胞发生失巢凋亡。更多还原

【Abstract】 Objective To investigate the effect and mechanisms of tumor suppressor gene PTEN on the induction of anoikis of human bladder transitional carcinoma cells BIU-87.Methods BIU-87 cells were transfected with GFP plasmids containing wild-type PTEN or mutant PTEN gene(G129E-PTEN,C124A-PTEN) in vitro.The PTEN expression and the phosphorylation levels of focal adhesion kinase(FAK) and protein kinase B(PKB/Akt) were detected by Western blot.Flow cytometry assay and laser scanning confocal microscopy were used to analyze apoptosis in adherent and non-adherent cells.Results Compared with the control,PTEN expression in the cells transfected with wild-type PTEN was increased to 210%-260%,while the phosphorylation level of FAK and Akt decreased by 59%(P<0.01) and 89%(P<0.01),respectively.The anoikis rate was increased from(8.32±0.57)% to(37.62±2.12)%.In the cells transfected with G129E-PTEN,the phosphorylation level of FAK and Akt was decreased by 62%(P<0.01) and 46%(P<0.05),respectively.The anoikis rate was increased by(32.57±1.72)%.In the cells transfected with C124A-PTEN,neither the phosphorylation of FAK and Akt nor the anoikis rate had obvious change,although the PTEN expression was enhanced remarkably as compared with the control group.Conclusion Through its phosphatase activity,tumor suppressor gene PTEN can suppress the phosphorylation of FAK and Akt,and induce anoikis in human bladder transitional carcinoma cells BIU-87.更多还原