摘要
目的探讨腺病毒介导的短发夹RNA(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)表达对体外活化肝星状细胞(HSC)p130Crk相关底物蛋白(p130Cas)及桩蛋白信号转导的影响。方法体外培养活化大鼠肝星状细胞系HSC-T6,以腺病毒为载体,将靶向PTEN的shRNA瞬时转染体外活化HSC,构建体外活化HSC的PTEN低表达模型;采用Western blot及实时荧光定量PCR技术检测活化HSC的PTEN、p130Cas及桩蛋白的蛋白及mRNA表达。实验分组:对照组:以DMEM代替腺病毒液转染HSC;Ad-绿色荧光蛋白(GFP)组:转染仅表达GFP的空病毒Ad-GFP;Ad-shRNA/PTEN组:转染携带靶向PTEN的shRNA并表达GFP的重组腺病毒Ad-shRNA/PTEN。多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体外活化HSC的PTEN蛋白及mRNA表达(P<0.05),体外活化HSC的PTEN低表达模型成功构建。3组HSC的P130Cas RNA表达比较,以对照组HSC的P130Cas mRNA表达量为1,则Ad-GFP组和Ad-shRNA/PTEN组HSC的P130Cas mRNA相对对照组的表达倍数分别为1.01倍、1.52倍,Ad-shRNA/PTEN组HSC的P130Cas mRNA表达明显高于对照组及Ad-GFP组(P<0.05),而对照组与Ad-GFP组之间差异无统计学意义(P>0.05);3组HSC的p130Cas蛋白表达比较,Ad-shRNA/PTEN组(0.93±0.08)高于对照组(0.74±0.07)及Ad-GFP组(0.72±0.02),P<0.05,而Ad-GFP组与对照组之间差异无统计学意义(P>0.05)。3组HSC的桩蛋白mRNA表达比较,以对照组HSC的桩蛋白mRNA表达量为1,则Ad-GFP组及Ad-shRNA/PTEN组HSC的桩蛋白mRNA相对对照组的表达倍数分别为0.97倍、1.58倍,Ad-shRNA/PTEN组HSC的桩蛋白mRNA表达高于对照组及Ad-GFP组(P<0.05),对照组与Ad-GFP组之间差异无统计学意义(P>0.05);3组HSC的桩蛋白蛋白表达比较,Ad-shRNA/PTEN组(0.91±0.05)高于对照组(0.46±0.03)及Ad-GFP组(0.50±0.04),P<0.05,而对照组与Ad-GFP之间差异无统计学意义(P>0.05)。结论PTEN表达下调可显著增强体外活�
Objective To investigate the role of adenovirus-mediated short hairpin RNA(shRNA)in down-regulating the expression of phosphatase and tensin homologue deleted on chromosome ten(PTEN)on p130Crk-related substrates(p130Cas)and paxillin signal transduction to activate hepatic stellate cell(HSC)in vitro.Methods The rat hepatic stellate cell line,HSC-T6 was cultured and activated in vitro.The adenovirus was used as a vector to transiently transfect shRNA targeting PTEN to activate HSC in vitro,and then PTEN low expression model of activated HSC in vitro was established.Western blot and real-time fluorescence quantitative PCR were used to detect the protein and mRNA expression of PTEN,p130cas and paxillin in activated HSC.The experiment was divided into control group(HSC were transfected with DMEM medium instead of adenovirus),Ad-GFP group(HSC were infected with empty the adenovirus expressing green fluorescent protein(GFP)alone),and Ad-shRNA/PTEN group(HSC were infected with the recombinant adenovirus containing both shRNA targeting PTEN and GFP gene).One-way analysis of variance was used for comparison of multiple groups,and LSD test was used for inter-group comparison.Results shRNA targeting PTEN was successfully transfected and significantly down-regulated the PTEN protein and mRNA expression of HSC in vitro(P<0.05),and the PTEN low expression model of HSC in vitro was successfully constructed.Compared with the expression of p130cas mRNA in the three groups of HSC,the expression fold of p130cas mRNA in the Ad-GFP group and the Ad-shRNA/PTEN group was 1.01 times and 1.52 times,respectively.The expression of p130cas mRNA in HSC of the Ad-shRNA/PTEN group was significantly higher than control group and Ad-GFP group(P<0.05),but there was no statistically significant difference between the control group and the Ad-GFP group(P>0.05).The expression of p130cas protein in the three groups was higher than that in the control group(0.74±0.07)and the Ad-GFP group(0.72±0.02);P<0.05,but there was no statistically significant di
作者
郝礼森
张朋垒
刘博
章广玲
陈静
宋洁
张明婷
靳丽敏
Hao Lisen;Zhang Penglei;Liu Bo;Zhang Guangling;Chen Jing;Song Jie;Zhang Mingting;Jin Limin(Department of Gastroenterology,the Affiliated Hospital of North China University of Science and Technology,Tangshan 063000,China;The Basic Medical College of North China University of Science and Technology,Tangshan 063000,China;The Life Science College of North China University of Science and Technology,Tangshan 063000,China)
出处
《中华肝脏病杂志》
CAS
CSCD
北大核心
2019年第12期989-993,共5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)。
关键词
肝纤维化
肝星状细胞
桩蛋白
信号传导
Hepatic fibrosis
Hepatic stellate cells
Paxillin
Signal transduction
