PTEN过表达及其突变对体外活化肝星状细胞凋亡的影响

目的 探讨过表达的野生型PTEN及其突变体G129E对体外培养的活化肝星状细胞(HSC)增殖、凋亡的影响及其机制.方法 体外培养活化的HSC,以腺病毒为载体将野生型PTEN基因及其突变体G129E基因瞬时转染HSC;四甲基偶氮唑盐(MTT)法检测HSC增殖;末端转移酶标记技术(TUNEL)及流式细胞术测定HSC凋亡;Western印迹及实时荧光定量PCR方法 检测HSC PTEN表达;Western印迹测定HSC Bcl-2及Bax表达.结果 外源性野生型PTEN基因及G129E基因成功转染体外活化HSC,并引起HSC的Bax表达增加,Bcl-2表达下降(P<0.01).过表达的野生型PTEN及G129E明显抑制HSC增殖,在转染HSC后48 h、72 h的增殖抑制率分别为14.03%、23.12%和9.52%、12.63%.野生型PTEN基因及G129E基因转染HSC后72 h,HSC凋亡率均显著增加(P<0.01).在上述作用中野生型PTEN均明显强于其突变体G129E.结论 过表达的野生型PTEN及其突变体G129E通过降低Bcl-2/Bax途径诱导体外活化HSC凋亡,并抑制其增殖;并且,野生型PTEN的作用明显强于G129E.

Effects of PTEN over-expression and its mutant on the apoptosis of activated hepatic steilate cells in vitro

Objective To investigate the effects of over-expression of wild-type PTEN gene and its mutant (G129E) on apoptosis and proliferation of activated hepatic stellate cells (HSC) in vitro and its potential mechanisms. Methods The activated HSC cells were cultured in vitro and transfected with PTEN gene and G129E gene via adenoviral vector. The apoptosis of HSC was measured by MTT , and its proliferation was assessed by TUNEL and flow cytometry (FCM) . Western blotting and real-time fluorescent quantitation PCR were used to detect expression of PTEN in HSC. And the changes of Bcl-2 and Bax expression were tested by Western blotting. Results The wild type PTEN gene and G129E gene were successfully transducted into HSC, which resuted in elevated expression of Bax and reduced expression of Bcl-2 (P<0.01). After transduction, HSC proliferation was markedly inhibited with inhibitory rates of 14.03% and 23.12% at 48 and 72 hours in Ad-PTEN ,respectively, as well as 9.52% and 12.63% in Ad-G129E, respectively. Apoptotic rate of HSC exposed to Ad-PTEN or Ad-G129E for 72 hours increased significantly (P<0.01). Furthermore, wild type PTEN was more powerful than G129E for above-mentioned effects. Conclusions Over-expression of wild type PTEN and its mutant G129E can inhibit the proliferation of activated HSC, and induce HSC apoptosis through the Bcl-2/Bax pathway. In addition, the effect of wild type PTEN is more powerful than that of G129E.