CAJ | 학술논문

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.
목적 탐토과표체적야생형PTEN급기돌변체G129E대체외배양적활화간성상세포(HSC)증식、조망적영향급기궤제.방법 체외배양활화적HSC,이선병독위재체장야생형PTEN기인급기돌변체G129E기인순시전염HSC;사갑기우담서염(MTT)법검측HSC증식;말단전이매표기기술(TUNEL)급류식세포술측정HSC조망;Western인적급실시형광정량PCR방법 검측HSC PTEN표체;Western인적측정HSC Bcl-2급Bax표체.결과 외원성야생형PTEN기인급G129E기인성공전염체외활화HSC,병인기HSC적Bax표체증가,Bcl-2표체하강(P<0.01).과표체적야생형PTEN급G129E명현억제HSC증식,재전염HSC후48 h、72 h적증식억제솔분별위14.03%、23.12%화9.52%、12.63%.야생형PTEN기인급G129E기인전염HSC후72 h,HSC조망솔균현저증가(P<0.01).재상술작용중야생형PTEN균명현강우기돌변체G129E.결론 과표체적야생형PTEN급기돌변체G129E통과강저Bcl-2/Bax도경유도체외활화HSC조망,병억제기증식;병차,야생형PTEN적작용명현강우G129E.
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.