Effects of PTEN Overexpression and Mutation on FAK Signal Transduction in Activated Hepatic Stellate Cells

doi:10.3969/j.issn.1007-9572.2016.29.011

Abstract

Abstract: Objective　To investigate the overexpression of wild-type PTEN and its mutant G129E on focal adhesion kinase（FAK） signal transduction in activated hepatic stellate cells（HSC） in vitro.Methods　Using transient transfection technique，the wild-type PTEN gene and G129E gene were transduced into the cultured activated HSC mediated by adenoviral vector from December 2014 to June 2015.Cells were grouped as follows:control group，activated HSC were transfected with DMEM basal medium instead of adenovirus.Ad-GFP group，activated HSC were infected with adenovirus expressing green fluorescent protein（GFP）.Ad-PTEN group，activated HSC were infected with the recombinant adenovirus containing both wild type PTEN and GFP gene.Ad-G129E group，activated HSC were infected with adenovirus harboring both PTEN mutant G129E and GFP gene.The protein expression of PTEN,FAK and phosphorylated FAK〔p-FAK（Tyr397）〕 in activated HSC were detected by Western blotting.PTEN and FAK mRNA levels were detected by real-time fluorescent quantitation PCR.Results　At 48 hours after adenovirus transfection，there were statistically significant difference in the expressions of PTEN protein and mRNA in activated HSC among 4 groups（P<0.05）,the expressions of PTEN protein and mRNA in activated HSC in Ad-PTEN group and Ad-G129E group were higher than those in control group and Ad-GFP group（P<0.05）,there were no statistically significant difference in the expressions of PTEN protein and mRNA in activated HSC between control group and Ad-GFP group or Ad-PTEN group and Ad-G129E group（P>0.05）.There were no statistically significant difference in the expressions of FAK protein and mRNA in activated HSC among 4 groups（P>0.05）.There was statistically significant difference in the expression of p-FAK（Tyr397）in activated HSC among 4 groups（P<0.05）,the expression of p-FAK（Tyr397）in activated HSC in Ad-PTEN group and Ad-G129E group was lower than that in control group and Ad-GFP group（P<0.05）,but there were no statistically significant difference in the expression of p-FAK（Tyr397）in activated HSC between control group and Ad-GFP group or Ad-PTEN group and Ad-G129E group（P>0.05）.Conclusion　The overexpression of wild-type PTEN and its mutant G129E can negatively regulate FAK signaling transduction by inhibiting phosphorylation of FAK in activated HSC in vitro.

Key words: Liver fibrosis, Hepatic stellate cells, PTEN, FAK, Signal transduction

摘要： 目的　探讨过表达的野生型第10号染色体缺失的磷酸酶张力蛋白同源物基因（PTEN）及其突变体G129E（仅保留蛋白磷酸酶活性而丧失脂质磷酸酶活性）对活化肝星状细胞（HSC）黏着斑激酶（FAK）信号转导的影响。方法　2014年12月—2015年6月，利用瞬时转染技术，以腺病毒为载体将野生型PTEN及其突变体G129E转染到体外培养的活化HSC；实验分为4组：对照组：腺病毒转染时以DMEM基础培养基代替腺病毒；Ad-GFP组：转染表达绿色荧光蛋白（GFP）的载体空病毒Ad-GFP；Ad-PTEN组：转染携带野生型PTEN并表达GFP的重组腺病毒Ad-PTEN；Ad-G129E组：转染携带G129E并表达GFP的重组腺病毒Ad-G129E。应用Western blotting法检测活化HSC中PTEN、FAK、磷酸化FAK〔p-FAK（Tyr397）〕表达，实时荧光定量PCR法检测活化HSC中PTEN、FAK mRNA表达。结果　腺病毒转染活化HSC 48 h，4组活化HSC中PTEN及其mRNA表达比较，差异有统计学意义（P<0.05）；其中Ad-PTEN组和Ad-G129E组活化HSC中PTEN及其mRNA表达高于对照组和Ad-GFP组（P<0.05）；对照组与Ad-GFP组、Ad-PTEN组与Ad-G129E组活化HSC中PTEN及其mRNA表达比较，差异无统计学意义（P>0.05）。4组活化HSC中FAK及其mRNA表达比较，差异无统计学意义（P>0.05）。4组活化HSC中p-FAK（Tyr397）表达比较，差异有统计学意义（P<0.05）；其中Ad-PTEN组和Ad-G129E组活化HSC中p-FAK（Tyr397）表达低于对照组和Ad-GFP组（P<0.05）；对照组与Ad-GFP组、Ad-PTEN组与Ad-G129E组活化HSC中p-FAK（Tyr397）表达比较，差异无统计学意义（P>0.05）。结论　过表达的野生型PTEN及其突变体G129E均可通过抑制活化HSC的FAK磷酸化负性调控体外活化HSC的FAK信号转导。

关键词: 肝纤维化, 肝星状细胞, 第10号染色体缺失的磷酸酶张力蛋白同源物基因, 黏着斑激酶, 信号传导

WEI Yue, HAO Li-sen, REN Chang-zhen, ZHANG Guang-ling, CHEN Jing, WANG Jing, MO Yan-bo, ZHANG Ming-ting. Effects of PTEN Overexpression and Mutation on FAK Signal Transduction in Activated Hepatic Stellate Cells[J]. Chinese General Practice, 2016, 19(29): 3562-3566.

魏月, 郝礼森, 任昌镇, 章广玲, 陈静, 王静, 莫艳波, 张明婷. PTEN过表达及突变对活化肝星状细胞黏着斑激酶信号转导的影响研究[J]. 中国全科医学, 2016, 19(29): 3562-3566.

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