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

发布时间：2019-06-06 06:06

【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(P0.05);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(P0.05),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(P0.05),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。
[Abstract]:Aim: to investigate the effect of adenoviral mediated shRNA down-regulation of phosphatase tensin homologue (PTEN) gene expression on (HSC) filamentous actin (F-actin) in activated hepatic stellate cells cultured in vitro. Methods: activated rat HSC (HSCT6) was cultured in vitro. Recombinant adenoviral AdshRNA/PTEN carrying PTEN targeting RNA interference sequence [short hairpin RNA (shRNA)] and expressing green fluorescent protein (GFP) and control empty virus Ad-GFP expressing GFP only were transfected into HSC,. The PTEN mRNA and protein expression of HSC were detected by real-time fluorescence quantitative PCR and Western blotting. The morphology, F-actin distribution, fluorescence intensity, pseudopod and stress fiber of HSC were detected by laser scanning confocal microscope, and the concentration of Ca~ (2) in HSC was detected by calcium fluorescence probe Rhod-2/AM. The experiment was divided into control (control) group (DMEM instead of adenoviral solution in adenoviral transfer step), Ad-GFP group (empty virus Ad-GFP expressing GFP) and Ad-shRNA/PTEN group (recombinant adenoviral Ad-shRNA/PTEN). Results: shRNA targeting PTEN was successfully transformed into activated HSC, in vitro to significantly down-regulate the expression of PTEN mRNA and protein in HSC (P 0.05). The down-regulation of PTEN expression made the activated HSC stretch around the star, the arrangement of F-actin was closely regular, the number increased, the pseudopod fully extended outward, and the stress filament increased and thickened. The fluorescence intensity of F-actin in Ad-shRNA/PTEN group was significantly higher than that in control group and Ad-GFP group (P 0.05), but there was no significant difference between control group and Ad-GFP group. The concentration of Ca~ (2) in HSC in Ad-shRNA/PTEN group was significantly higher than that in control group and Ad-GFP group (P 0.05), but there was no significant difference between control group and Ad-GFP group. Conclusion: the down-regulation of PTEN expression enhances the formation of activated hepatic stellate cytoskeleton protein F-actin and the remodeling of cytoskeleton in vitro, and increases the concentration of Ca~2 in HSC.
【作者单位】：华北理工大学附属医院消化内科;华北理工大学基础医学院;
【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)
【分类号】：R575.2

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