摘要
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)刺激的肝星状细胞(HSC),探讨膜型基质金属蛋白酶1(MT1-MMP)在FRNK诱导HSC凋亡中的作用。方法在体外,以FN刺激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和透射电镜技术检测细胞的凋亡,蛋白免疫印迹及RT-PCR方法检测FRNK、FAK、p-FAK(Tyr397)、MT1-MMP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HSC 48小时后,HSC凋亡率由(9.28±1.05)%增加至(25.37±1.92)%(P<0.01)。FRNK抑制FAK磷酸化后在翻译和转录水平上调MT1-MMP表达,2.26±0.14 vs 1.09±0.15(P<0.01);1.58±0.18 vs1.00±0.10(P<0.01)。结论在脂质体介导下瞬时转染FRNK表达质粒可诱导HSC发生凋亡,上调MT1-MMP可能是其机制之一。
Objective To investigate the effect of breaking the phosphorylation of focal adhesion kinase（FAK） by FAK related non-kinase（FRNK） on the apoptosis and the expression of membrane-type matrix metalloproteinase-1 （MT1-MMP） in hepatic stellate cell（HSC） in vitro. Methods After fibroneetin（FN） stimulated HSC,FRNK plasmid mediated by cationic liposome was transfeeted into HSC. The apoptosis of FRNK-indueed HSC was examined by annexin-V/propidium iodide double-labeled flow cytometry （FCM）, gel electrophoresis and transmission electron microscope. And the levels of FRNK,FAK,p-FAK（Tyr397） and MT1-MMP in HSC were assayed by Western blot on protein level, and by RT-PCR on mRNA level, respectively. Results The expression of FRNK was enhanced after FRNK had been transiently transfected into HSC in vitro. The apoptotic rate in HSC exposed to FRNK plasmid for 48 hours was higher than that in non-FRNK plasmid group,（25.37±1.92） % vs （9.28±1.05）% （PG0.01）,and accompanied by a significant increase of MT1-MMP activity both in the protein and in the mRNA level,2.26±0.14 vs 1.09±0.15 （ P G0.01） ; 1.58±0.18 vs 1.00±0.10 （ P 〈0.01）. Conclusion FRNK can induce HSC apoptosis and MT1-MMP is perhaps involved in the process.
出处
《临床荟萃》
CAS
2009年第15期1309-1312,共4页
Clinical Focus
基金
河北省自然科学基金(C2008001133)
河北省卫生厅资助项目(2003055)
关键词
肝硬化
细胞凋亡
膜蛋白质类
liver cirrhosis
apoptosis
membrane protein
