腺病毒介导的shRNA下调PTEN表达对活化肝星状细胞骨架蛋白F-actin的影响  

Effects of adenovirus-mediated shRNA targeting PTEN on cytoskeletal protein F-actin in activated hepatic stellate cells

作　　者：郝礼森 宋小杰 章广玲 王静 刘博 张朋垒 张明婷 靳丽敏 

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：《中国病理生理杂志》2017年第3期557-561,566,共6页Chinese Journal of Pathophysiology

基　　金：河北省自然科学基金资助项目(No.H2013209327);中国肝炎防治基金会天晴肝病研究基金资助项目(No.CFHPC20132078)

摘　　要：目的:探讨腺病毒介导的shRNA下调第10号染色体缺失的磷酸酶张力蛋白同源物(PTEN)基因表达对体外培养的活化肝星状细胞(HSC)纤丝状肌动蛋白(F-actin)的影响。方法:体外培养大鼠活化HSC(HSCT6),将携带靶向PTEN的RNA干扰序列[短发夹RNA(shRNA)]并表达绿色荧光蛋白(GFP)的重组腺病毒AdshRNA/PTEN及仅表达GFP的对照空病毒Ad-GFP转染HSC,实时荧光定量PCR及Western blotting实验检测HSC的PTEN mRNA及蛋白表达;利用激光扫描共聚焦显微镜检测HSC的形态、F-actin的分布及荧光强度、伪足以及应力纤维的变化,并采用钙荧光探针Rhod-2/AM负载检测HSC内Ca^(2+)浓度的变化。实验分为对照(control)组(在腺病毒转染步骤以DMEM代替腺病毒液)、Ad-GFP组(转染表达GFP的空病毒Ad-GFP)和Ad-shRNA/PTEN组(转染重组腺病毒Ad-shRNA/PTEN)。结果:靶向PTEN的shRNA成功转染体外活化HSC,显著下调HSC的PTEN mRNA及蛋白表达(P<0.05);PTEN表达下调使活化HSC呈星形向四周伸展,F-actin排列紧密规则,数量增多,伪足充分向外伸展,应力纤维丝增长增粗;Ad-shRNA/PTEN组F-actin的荧光强度较control组及Ad-GFP组显著增强(P<0.05),而control组与Ad-GFP组间差异无统计学显著性;Ad-shRNA/PTEN组HSC内Ca^(2+)浓度较control组及Ad-GFP组明显升高(P<0.05),而control组与Ad-GFP组间差异无统计学显著性。结论:PTEN表达下调使体外活化肝星状细胞骨架蛋白F-actin的形成及细胞骨架的重构增强,并增加了HSC内的Ca^2浓度。AIM：To investigate the effects of down-regulation of phosphatase and tensin homology detected on chromosome 10 （ PTEN） gene by adenovirus-mediated short hairpin RNA （ shRNA） on cytoskeletal protein filamentous ac-tin （F-actin） in activated hepatic stellate cells （HSC） in vitro.METHODS： The activated HSC （HSC-T6 cells） were cultured in vitro and transfected with recombinant adenovirus carrying shRNA targeting PTEN and expressing green fluores -cent protein （ GFP）, Ad-shRNA/PTEN, and the control adenovirus expressing GFP only , Ad-GFP.The expression of PTEN in the HSC was measured by Western blotting and real-time fluorescent quantitative PCR （RT-qPCR）.Under the la-ser scanning confocal microscope , the cellular morphology , distribution and fluorescence intensity of F-actin, stress fibers and pseudopodia in activated HSC were examined by phalloidin marked with tetramethylrhodamine isothiocyanate （ TRITC） .The concentration of intracellular Ca 2＋in the HSC was detected by calcium fluorescent probe Rhod-2/AM.The HSC were divided into control group （ using DMEM instead of adenovirus for transfection ） , Ad-GFP group （ the HSC were transfected with the empty adenovirus expressing GFP alone ） and Ad-shRNA/PTEN group （ the HSC were transfected with recombinant adenovirus Ad-shRNA/PTEN） .RESULTS：The shRNA targeting PTEN was successfully transfected into ac-tivated HSC in vitro, and significantly down-regulated the expressions of PTEN at protein and mRNA levels in the HSC （P〈0.05）.The HSC with PTEN knockdown showed starlike and stretched to the surrounding .The F-actin of the cells was closely arranged in order and had the increasing number .The pseudopodia fully extended outward , and stress fibers got larger and thicker .The fluorescence intensity of F-actin in Ad-shRNA/PTEN group significantly enhanced （ P〈0.05 ） compared with control group and Ad-GFP group, while no significant difference between control group and Ad-GFP group was observed .Intracellular

关 键 词：PTEN RNA干扰 肝星状细胞 细胞骨架 纤丝状肌动蛋白 

分 类 号：R363.2[医药卫生—病理学]