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

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

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。

Objective To investigate the overexpression of wild- type PTEN and its mutant G129 E 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 G129 E 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-G129 E group,activated HSC were infected with adenovirus harboring both PTEN mutant G129 E 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-G129 E 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-G129 E 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-G129 E 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-G129 E group（ P〈0. 05）. Conclusion The overexpression of wild- type PTEN and its mutant G129 E can negatively regulate FAK signaling transduction by inhibiting phosphorylation of FAK in activated HSC in vitro.