肿瘤抑制基因PTEN对体外活化肝星状细胞骨架蛋白vinculin的影响  

Effects of tumor suppressor gene PTEN on the cytoskeletal protein vinculin in activated hepatic stellate cells in vitro

作　　者：郝礼森 张明婷 王玉兰 宋小杰 宋洁 章广玲 莫艳波 靳丽敏 张朋垒 HAO Lisen;ZHANG Mingting;WANG Yulan;SONG Xiaojie;SONG Jie;Zhang Guangling;MO Yanbo;JIN Limin;ZHANG Penglei(The Affiliated Hospital of North China University of Science and Technology,Tangshan 063000,China)

机构地区：[1]华北理工大学附属医院消化内科,河北唐山063000 [2]华北理工大学基础医学院,河北唐山063000

出　　处：《实用医学杂志》2018年第14期2298-2302,共5页The Journal of Practical Medicine

基　　金：河北省自然科学基金资助项目(编号:H2013209327);中国肝炎防治基金会天晴肝病研究基金资助项目(编号:CFH-PC20132078)

摘　　要：目的探讨第10号染色体缺失的磷酸酶张力蛋白同源物基因(PTEN)表达变化对体外活化肝星状细胞(HSC)纽蛋白(vinculin)的影响。方法分别将含有野生型PTEN基因及含有靶向PTEN的RNA干扰序列短发夹RNA(shRNA)的重组腺病毒转染体外培养的活化大鼠肝星状细胞系HSC-T6,构建体外活化大鼠肝星状细胞PTEN过表达及低表达模型;应用免疫荧光法并结合激光扫描共聚焦显微镜技术检测活化HSC的vinculin分布及表达。实验分组:(1)Control组:以无血清无抗生素的DMEM培养液代替腺病毒液转染体外活化HSC;(2)Ad-GFP组:以仅带有绿色荧光蛋白(GFP)基因的载体空病毒Ad-GFP转染体外活化HSC;(3)Ad-PTEN组:以带有GFP基因并携带野生型PTEN基因的重组腺病毒Ad-PTEN转染体外活化HSC;(4)Ad-shRNA/PTEN组:以带有GFP基因并携带靶向PTEN的shRNA的重组腺病毒Ad-shRNA/PTEN转染体外活化HSC。结果外源性野生型PTEN基因及靶向PTEN的shRNA成功转染体外活化HSC,并成功构建体外活化肝星状细胞PTEN过表达及低表达模型。HSC的vinculin蛋白主要表达于胞浆,4组HSC的vinculin亚细胞分布未发生明显变化。Ad-PTEN组HSC的vinculin蛋白荧光强度(251.78±12.68)明显低于Control组(381.13±9.12)及Ad-GFP组(383.87±12.15),P<0.05;而Ad-shRNA/PTEN组HSC的vinculin蛋白荧光强度(770.77±20.12)显著高于Control组及Ad-GFP组(P<0.05);但Control组与Ad-GFP组之间HSC的vinculin蛋白荧光强度差异无统计学意义(P>0.05)。结论 PTEN表达变化对体外活化肝星状细胞骨架蛋白vinculin的亚细胞分布无明显影响,但PTEN过表达可下调体外活化肝星状细胞骨架蛋白vinculin的表达,而PTEN低表达则可上调体外活化肝星状细胞骨架蛋白vinculin的表达。Objective To investigate the effects of over-expression and lower-expression of phosphatase and tension homology of chromosome ten(PTEN)on cytoskeletal protein vinculin in activated hepatic stellate cells(HSC)in vitro.Methods Through adenovirus vector,the wild PTEN gene or short hairpin RNA(shRNA)target-ing PTEN were transfected into cultural activated rat HSC(HSC-T6)in vitro respectively.The expressions of PTEN mRNA were measured by real time fluorescent quantitative PCR.Under laser scanning confocal microscope,the expression and location of vinculin in the activated HSC were analyzed by immunofluorescence.The experiment groups were as follow:control group,HSC were transfected with DMED instead of adenovirus.Ad-GFP group,HSC were transfected with empty adenovirus expressing green fluorescent protein(GFP)only.Ad-PTEN group,HSC were transfected with recombinant adenovirus harboring both GFP and wild PTEN gene.Ad-shRNA/PTEN group,HSC were transfected with recombinant adenovirus expressing both shRNA targeting PTEN and GFP.Results The wild PTEN gene and shRNA targeting PTEN were successfully transfected into activated HSC in vitro respectively,and the cellular model of over-expression and lower-expression of PTEN were successfully constructed too.The vinculin mostly distributed in the cytoplasm of HSC,and no significant changes were discovered in the subcellular distribution of vinculin in HSC among four groups.The fluorescence intensity of vinculin in Ad-PTEN group(251.78±12.68)was significantly(P<0.05)decreased,compared with control group(381.13±9.12)and Ad-GFP group(383.87±12.15),and the fluorescence intensity of vinculin in Ad-shRNA/PTEN group(770.77±20.12)was magnificently(P<0.05)higher than that of control group and Ad-GFP group,while no significant differences between control group and Ad-GFP group were discovered(P>0.05).Conclusions The over-expression of PTEN significantly decreases the expression of cytoskeletal protein vinculin,while the down-regulation of PTEN expression raises the expression of cyto

关 键 词：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 细胞骨架蛋白 纽蛋白 

分 类 号：R575.2[医药卫生—消化系统]