腺病毒介导的shRNA下调SHP2表达对人肝星状细胞LX-2凋亡的影响  

Effect of adenovirus-mediated shRNA down-regulates SHP2 expression on the apoptosis of human hepatic stellate cells LX-2

作　　者：郝礼森 展宗媛 宋洁 苗笑佳 何宇 蒋美钰 季景秀 莫艳波 Hao Lisen;Zhan Zongyuan;Song Jie;Miao Xiaojia;He Yu;Jiang Meiyu;Ji Jingxiu;Mo Yanbo(Department of Gastroenterology,The Affiliated Hospital of North China University of Science and Technology,Tangshan 063000,China)

机构地区：[1]华北理工大学附属医院消化内科,唐山063000

出　　处：《中华肝脏病杂志》2023年第12期1313-1317,共5页Chinese Journal of Hepatology

基　　金：河北省自然科学基金面上项目(H2018209366)。

摘　　要：目的探讨腺病毒介导的短发夹RNA(shRNA)下调含SH2结构域的蛋白酪氨酸磷酸酶2(SHP2)对体外培养的人肝星状细胞LX-2凋亡的影响。方法将携带靶向SHP2的shRNA并表达绿色荧光蛋白(GFP)的重组腺病毒Ad-shRNA/SHP2及仅表达GFP的对照空病毒Ad-GFP分别转染体外培养的LX-2细胞;实时荧光定量PCR检测LX-2细胞的SHP2 mRNA表达;用Western blot检测LX-2细胞的SHP2、Bax及Bcl-2蛋白表达;TUNEL及膜联蛋白-V/碘化丙啶双标记流式细胞术检测LX-2细胞凋亡。实验分组:(1)对照组:以DMEM代替腺病毒转染LX-2细胞;(2)Ad-GFP组:转染空病毒Ad-GFP;(3)Ad-shRNA/SHP2组:转染重组腺病毒Ad-shRNA/SHP2。多组间均数比较用单因素方差分析,组间比较采用LSD检验。结果靶向SHP2的shRNA显著下调LX-2细胞的SHP2蛋白及mRNA表达(P<0.05);TUNEL及膜联蛋白-V/碘化丙啶双标记流式细胞术检测结果显示,与对照组LX-2细胞凋亡率(3.077%±0.731%,9.438%±0.804%)及Ad-GFP组(3.250%±0.851%,8.893%±1.982%)比较,Ad-shRNA/SHP2组LX-2细胞凋亡率(12.755%±1.606%,19.340%±2.505%)明显升高(P<0.05),而对照组与Ad-GFP组之间差异无统计学意义(P>0.05)。Western blot检测各组LX-2细胞的Bax及Bcl-2蛋白表达结果显示,与对照组及Ad-GFP组LX-2细胞的Bax蛋白表达(1.989±0.147,1.999±0.162)比较,Ad-shRNA/SHP2组LX-2细胞Bax蛋白表达(2.493±0.203)显著升高(P<0.05),而对照组与Ad-GFP组之间差异无统计学意义(P>0.05);与对照组及Ad-GFP组LX-2细胞的Bcl-2蛋白表达(1.707±0.146,1.521±0.142)比较,Ad-shRNA/SHP2组LX-2细胞Bcl-2蛋白表达(1.042±0.148)明显降低(P<0.05),而对照组与Ad-GFP组之间差异无统计学意义(P>0.05)。结论SHP2表达下调通过降低Bcl-2/Bax诱导体外人肝星状细胞LX-2凋亡。Objective To investigate the effect of adenovirus-mediated short hairpin RNA(shRNA)downregulating SH2 domain-containing protein tyrosine phosphatase 2(SHP2)on the apoptosis of human hepatic stellate cells LX-2 cultured in vitro.Methods The recombinant adenovirus Ad-shRNA/SHP2 carrying shRNA targeted SHP2 and expressing green fluorescent protein(GFP),and the empty control virus Ad-GFP expressing GFP were transfected into LX-2 cells cultured in vitro.Real-time fluorescence quantitative PCR was used to detect SHP2 mRNA expression in LX-2 cells.Western blot was used to detect the protein expressions of SHP2,Bax,and Bcl-2 in LX-2 cells.TUNEL and annexin-V/propidium iodide dual-labeled flow cytometry were used to detect apoptosis in LX-2 cells.Experimental group:(1)Control group:LX-2 cells were transfected with DMEM instead of adenovirus;(2)Ad-GFP group:transfected with empty virus Ad-GFP;(3)Ad-shRNA/SHP2 group:transfected with recombinant adenovirus Ad-shRNA/SHP2.The means between multiple groups were compared using a one-way ANOVA and the LSD test was used for inter group comparisons.Results shRNA-targeted SHP2 significantly down-regulated the expression of SHP2 protein and mRNA in LX-2 cells(P<0.05).The TUNEL and annexin-V/propidium iodide dual-labeled flow cytometry results showed that the apoptosis rate of LX-2 cells in the Ad-shRNA/SHP2 group(12.755%±1.606%,19.340%±2.505%)(P<0.05)was significantly higher compared to the control group(3.077%±0.731%,9.438%±0.804%)and the Ad-GFP group(3.250%±0.851%,8.893%±1.982%),with no statistically significant difference between the control group and the Ad-GFP group(P>0.05).Western blot analysis of Bax and Bcl-2 protein expression in LX-2 cells of each group revealed that the Bax protein expression was significantly higher in the Ad shRNA/SHP2 group(2.493±0.203)(P<0.05)compared to the control group and Ad-GFP group(1.989±0.147,1.999±0.162),with no statistically significant difference between the control group and the Ad-GFP group(P>0.05),while the Bcl-2 protein was sign

关 键 词：细胞凋亡 肝星状细胞 SHP2 

分 类 号：R575[医药卫生—消化系统]