黏着斑激酶相关非激酶对肝星状细胞凋亡及细胞外信号调节激酶的影响

目的 应用黏着斑激酶相关非激酶(FRNK)表达质粒瞬时转染纤维连接蛋白(FN)预刺激的肝星状细胞(HSC),探讨FRNK对HSC凋亡及细胞外信号调节激酶(ERK)的影响.方法 在体外,以FN诱导HSC增殖,采用脂质体介导的方法用FRNK表达质粒瞬时转染HSC,应用膜联蛋白/碘化丙啶双标记流式细胞术、DNA凝胶电泳技术和透射电镜技术检测细胞的凋亡,Western blot及RT-PCR方法检测FRNK、黏着斑激酶(FAK),p FAK(Tyr397)、半胱氨酸天冬氨酸特异性蛋白酶-3(caspase-3)、ERK1、p-ERK蛋白及其mRNA表达. 结果FRNK表达质粒成功转染HSC,在翻译后水平抑制FAK磷酸化.与空质粒组比较,FRNK表达质粒转染HSC48 h后,HSC凋亡率由9.28%±1.05%增至25.37%±1.92%(P<0.01),caspase-3蛋白由185.82±9.69增至264.17±12.60(P<0.01),caspase-3 mRNA由1.07±0.27增至4.19±0.48(P<0.01).FRNK抑制FAK磷酸化和在翻译和转录水平抑制ERK1、p-ERK的表达,而FN则促进FAK和ERK1,p-ERK在翻译和转录水平的表达. 结论在脂质体介导下瞬时转染FRNK表达质粒,可使外源性的FRNK在HSC内大量表达,在翻译后水平抑制FAK磷酸化;并可能通过FAK-ERK信号转导通路诱导FN刺激的HSC发生凋亡.

The role of FAK-ERK signal transduction pathway in apoptosis of hepatic stellate cell

Objectives To investigate the effects of FAK related non-kinase(FRNK)on the apoptosis of hepatic stellate cells(HSC)in vitro and on the extracellular signal-regulated kinase(ERK)signal transduc-tion pathway．Methods HSC were stimulated by fibronectin(FN)，and then they were transfected with FAK-related non-kinase(FRNK) plasmids mediated by cationic liposome．The apoptosis of FRNK-induced HSC was examined by annexin-V／propidium iodide double-labeled flow cytometry(FCM)，gel electrophoresis and transmission electron microscopy．Levels of FRNK，FAK，p-FAK(Tyr397)，ERKl and P-ERK in HSC were assayed bv Western blot on the protein level，and by RT-PCR on the mRNA level．Results The expression of FRNK was enhanced after FRNK plasmids were transiently transfected into the HSC．The apoptotic rate of the HSC exposed to FRNK plasmids for 48 h was higher than that in the non-FRNK plasmid group(25．37％±1．92％vs 9．28％±1．05％，P<0．01)，and was accompanied by a significantly higher activity of caspase-3 both in the protein and in the mRNA levels(264．17±12．60 vs 185．82±9．69)，P<0．01;(4．19±0．48 vs 1．07±0．27)，P<0．01]．After exposure of HSC to FRNK plasmids，compared with the non-FRNK plasmid group，the expressions of p-FAK，ERK1 and p-ERK in protein and mRNA levels were lower；on the contrary,compared with the control group，the expressions of p-FAK，ERKl and p-ERK in the FN group were higher．Conclusions The expression of FRNK was enhanced and the phosphorylation of FAK was inhibited after FRNK was transiently transfected into HSC in vitro. FRNK induces apoptosis of HSC. FAK-ERK signal transduction pathway perhaps is involved in the process.