FRNK质粒转染抑制肝星状细胞增殖机制研究  

FAK-RELATED NON-KINASE PLASMID TRANSFECTION INHIBITED HEPATIC STELLATE CELLS PROLIFERATION

作　　者：霍晓霞[1,2] 张晓岚[1] 申建刚[1] 魏娟[1] 窦永青[3] 

机构地区：[1]河北医科大学第二医院消化科,河北石家庄050000 [2]石家庄市第三医院消化科,河北石家庄050000 [3]石家庄卫生学校,河北石家庄050000

出　　处：《中国应用生理学杂志》2009年第1期69-73,共5页Chinese Journal of Applied Physiology

基　　金：河北省自然科学基金资助项目(301361);河北省卫生厅资助项目(2003055)

摘　　要：目的:探讨黏着斑激酶相关非激酶(FRNK)质粒瞬时转染肝星状细胞(HSC),对纤维连接蛋白(FN)刺激的HSC增殖的影响及其机制。方法:应用体外细胞培养技术,脂质体介导法进行FRNK质粒转染;Western blot技术检测FRNK、黏着斑激酶(FAK)、p-FAK(Tyr^(397))蛋白的表达情况,鉴定转染效果;改良MTT法测定HSC增殖;流式细胞术(FCM)检测细胞周期时相。结果:FRNK质粒转染HSC 48 h时,FRNK蛋白表达最强(P<0.01);FAK蛋白转染前后无明显差异(P>0.01);FRNK质粒组p-FAK(Tyr^(397))蛋白含量(0.40±0.14)较空质粒组(1.89±0.25)显著下降(P<0.01);FRNK在HSC大量表达后,于转染12 h、24 h、48 h的HSC增殖抑制率分别为20.07%、26.16%和29.77%(P<0.01),呈时间依赖性关系;FRNK质粒组G0/G1期细胞数(71.4±2.81)较空质粒组(48.9±1.66)明显增加(P<0.01)。结论:脂质体介导下FRNK质粒转染,可使外源性FRNK在HSC内大量表达,抑制FAK磷酸化,阻滞HSC周期时相于G0/G1期,抑制HSC增殖。Aim： To observe the effect of FAK-related non-Kinase（FRNK） plasmid on hepatic stellate cell（HSC） proliferation stimulated by fibronectin（FN）. Methods： FRNK plasmid was transfeeted into HSC with transient liposomal transfeetion. The proteins of FRNK, FAK and p-FAK（Tyr^397） were assayed by Western blotting analysis. The prohferation of HSC was evaluated by improved MTT assay, and cell cycle pattern was determind by flow cytometry（FCM）. Results： （1）The expression of FRNK protein increased after FRNK transfected HSC, and it was at 48 h that the expression of FRNK protein was the highest（ P 〈 0.01）. The protein level of FAK was no significant difference between before FRNK plamid transfection and after transfection（ P 〉 0.05）. The expression of p-FAK（Tyr^397） protein was down-regulated after FRNK had been transfected in HSC, （ P 〈 0.01 ）. （2） The HSC proliferation inhibition rates at 12 h, 24 h and 48 h after FRNK transfection were 20.07 %, 26.16 %, 29.77 %, respectively（ P 〈 0.01）. （3）Compared with the non-FRNK plasmid group, the FRNK-transfected HSCs almost arrested in G0/G1 phase（71.4 ± 2.81 vs 48.9 ±1.66, P 〈 0.01）. Conclusion： After FRNK were transfected successfully in HSCs using lipofectamine, the phosphorylation of FAK was inhibited. The HSC proliferation was restrained in a time-dependent manner and the HSC was arrested in G0/G1 phase.

关 键 词：肝星状细胞 增殖 黏着宽激酶相关非激酶 黏着斑激酶 细胞周期 

分 类 号：R575[医药卫生—消化系统]