学术论文

      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

      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(HSC-T6),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Ad-shRNA/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Ca2+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 mR-NA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Ca2+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Ca2浓度.
      Abstract:
      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 Ca 2+concentration of the HSC in Ad-shRNA/PTEN group was statistically higher ( P<0.05 ) than that in control group and Ad-GFP group, and no significant difference between control group and Ad-GFP group was found.CONCLUSION:Down-regulation of PTEN expression promotes the formation of F-actin and reorganization of cy-toskeleton in activated HSC , and increases the intracellular Ca 2+concentration in activated HSC in vitro.
      作者: 郝礼森 [1] 宋小杰 [1] 章广玲 [2] 王静 [1] 刘博 [1] 张朋垒 [1] 张明婷 [1] 靳丽敏 [1]
      作者单位: 华北理工大学 附属医院消化内科,河北唐山,063000 华北理工大学 基础医学院,河北唐山,063000
      刊 名: 中国病理生理杂志 ISTICPKU
      年,卷(期): 2017, 33(3)
      分类号: R363.2
      在线出版日期: 2017年4月10日
      基金项目: 河北省自然科学基金资助项目,中国肝炎防治基金会天晴肝病研究基金资助项目