腺病毒介导的shRNA下调PTEN表达对活化肝星状细胞微丝结合蛋白vinculin、filamin A及cortactin的影响  

Effects of adenovirus-mediated shRNA down-regulates PTEN expression on fibril-binding proteins vinculin,filamin A and cortactin in activated hepatic stellate cells

作　　者：郝礼森 宋洁 张明婷 宋小杰 蒋美钰 季景秀 莫艳波 王静 Hao Lisen;Song Jie;Zhang Mingting;Song Xiaojie;Jiang Meiyu;Ji Jingxiu;Mo Yanbo;Wang Jing(Department of Gastroenterology,the Affiliated Hospital of North China University of Science and Technology,Tangshan 063000,China;The First Hospital of Zhangjiakou City,Zhangjiakou 075000,China;The People's Hospital ofCangzhou City,Cangzhou 061000,China)

机构地区：[1]华北理工大学附属医院消化内科,唐山063000 [2]张家口市第一医院,张家口075000 [3]沧州市人民医院,沧州061000

出　　处：《中华肝脏病杂志》2022年第1期38-44,共7页Chinese Journal of Hepatology

基　　金：河北省自然科学基金(H2013209327);中国肝炎防治基金会天晴肝病研究基金(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基因(PTEN)表达对活化肝星状细胞(HSC)的纽蛋白(vinculin)、细丝蛋白A(filamin A)及皮层肌动蛋白(cortactin)的影响。方法体外培养活化大鼠肝星状细胞系HSC-T6,将携带靶向PTEN的RNA干扰序列[短发夹RNA(short hairpin RNA,shRNA)]的重组腺病毒Ad-shRNA/PTEN及对照空病毒Ad-GFP转染HSC;采用实时荧光定量PCR及Western blot技术检测各组HSC的PTEN mRNA及蛋白表达;借助激光扫描共聚焦显微镜,采用免疫荧光法检测各组HSC的vinculin、filamin A及cortactin表达变化,并利用Image-pro plus 6.0软件进行图像分析处理,计算所测蛋白荧光表达的积分光密度值(IOD)。实验分为3组:对照组(在腺病毒转染步骤以DMEM代替腺病毒液)、Ad-GFP组(转染仅表达绿色荧光蛋白的空病毒Ad-GFP)、Ad-shRNA/PTEN组(转染携带靶向PTEN的shRNA并表达绿色荧光蛋白的重组腺病毒Ad-shRNA/PTEN)。3组间均数比较采用单因素方差分析,组间比较采用LSD检验。结果靶向PTEN的shRNA成功转染并显著下调HSC的PTEN mRNA及蛋白表达(P<0.05);HSC的vinculin主要表达于细胞质,Ad-shRNA/PTEN组HSC的vinculin荧光IOD(19758.83±1520.60)较对照组(7737.16±279.93)及Ad-GFP组(7725.50±373.03)显著升高(P<0.05),而对照组与Ad-GFP组之间HSC的vinculin荧光IOD差异无统计学意义(P>0.05)。3组HSC的filamin A荧光IOD差异无统计学意义(P>0.05),但3组HSC的filamin A亚细胞分布发生了变化,Ad-shRNA/PTEN组HSC的filamin A主要分布于细胞质,而对照组和Ad-GFP组HSC的filamin A主要位于细胞核,Ad-shRNA/PTEN组HSC的filamin A核质比(0.60±0.15)明显低于对照组(1.20±0.15)及Ad-GFP组(1.08±0.23),P<0.05;而对照组与Ad-GFP组之间HSC的filamin A核质比差异无统计学意义(P>0.05)。3组HSC的cortactin主要分布于细胞质,Ad-shRNA/PTEN组HSC的cortactin荧光IOD(54688.50±2095.53)较对照组(22959.94±1710.42)及Ad-GFP组(22547.11±1588.72Objective To investigate the effect of adenovirus-mediated shRNA down-regulating phosphatase and tensin homolog deleted on chromosome 10(PTEN)expression on vinculin,filamin A,and cortactin in activated hepatic stellate cells(HSCs).Methods Activated rats hepatic stellate cell line(HSC-T6)was cultured in vitro.Recombinant adenovirus Ad-shRNA7PTEN carrying PTEN targeted RNA interference sequence[short hairpin RNA(shRNA)]and empty control virus Ad-GFP were transfected into HSCs.The PTEN mRNA and protein expression of HSCs in each group were detected by real-time fluorescence quantitative PCR and Western blot.The expressional change of vinculin,filamin A and cortactin in HSCs of each group were detected by confocal laser scanning immunofluorescence microscope.Image-pro plus 6.0 software was used for image analysis and processing.The integrated optical density(IOD)of the fluorescence protein expression was measured.The experiment was divided into three groups:control group(DMEM instead of adenovirus solution in the adenovirus transfection step),Ad-GFP group(transfected with empty virus Ad-GFP only expressing green fluorescent protein),and Ad-shRNA/PTEN group(recombinant adenovirus Ad-shRNA/PTEN carrying shRNA targeting PTEN and expressing green fluorescent protein).One-way analysis of variance was used for comparison of mean value among the three groups,and LSD-test was used for comparison between the groups.Results shRNA targeted PTEN was successfully transfected and the expression of PTEN mRNA and protein in HSC(P<0.05)was significantly down-regulated.HSCs vinculin was mainly expressed in the cytoplasm.HSCs vinculin fluorescence IOD in the Ad-shRNA/PTEN group(19758.83±1520.60)was higher than control(7737.16±279.93)and Ad-GFP group(7725.50±373.03)(P<0.05),but there was no statistically significant difference between control group and Ad-GFP group(P〉0.05).There was no statistically significant difference in the fluorescence IOD of Filamin A among the three groups(P>0.05),but the subcellular distribution of Filamin

关 键 词：星状细胞 细丝蛋白A 微丝结合蛋白 纽蛋白 皮层肌动蛋白 

分 类 号：R575.2[医药卫生—消化系统]