CCl4诱导的肝纤维化小鼠和HSC-T6细胞miR-122水平变化及其意义探讨  

Micro-122 levels in liver fibrosis in vitro and in vivo

作　　者：王燕[1] 李伟甲 李娅[1] 陈香宇[1] 徐峰 Wang Yan;Li Weijia;Li Ya(Department of Gastroenterology,First Affiliated Hospital,Zhengzhou University,Zhengzhou 450052,Henan Province,China)

机构地区：[1]郑州大学第一附属医院消化科,郑州市450052 [2]河南省医疗器械检验所

出　　处：《实用肝脏病杂志》2023年第1期19-22,共4页Journal of Practical Hepatology

基　　金：河南省医学科技攻关计划联合共建项目(编号:LHGJ20190287)

摘　　要：目的研究微小RNA(miR)-122在肝星状细胞活化/增殖及肝纤维化发生过程中的水平变化及其可能的作用机制.方法建立CCl_(4)诱导的C57BL/6小鼠肝纤维化模型,以10 ng/ml转化生长因子-β1(TGF-β1)处理HSC-T6细胞,分别在小鼠体内和肝星状细胞转染miR-122激动剂和miR-122模拟物以过表达miR-122.采用RT-PCR和Western-blot法检测组织和细胞miR-122、α-平滑肌肌动蛋白(α-SMA)、I型胶原、金属蛋白酶组织抑制因子1(TIMP-1)和血小板衍生生长因子(PDGF)表达,采用CCK-8法检测HSC-T6细胞增殖.结果模型组小鼠肝组织α-SMA表达水平显著高于对照组(9.92±2.12对1.12±0.54,P<0.01),而miR-122水平显著低于对照组(0.95±0.31对2.07±0.28,P<0.01);在CCl_(4)诱导的肝纤维化小鼠,miR-122激动剂转染组肝组织miR-122水平显著高于miR-122激动剂对照组(6.27±1.73对2.78±0.21,P<0.01);miR-122激动剂转染组肝组织α-SMA、I型胶原、TIMP-1和PDGF蛋白水平显著下降;在TGF-β1处理的HST-T6细胞,随着处理时间的延长,肝星状细胞α-SMA水平逐渐升高(0h:0.61±0.02,12 h:0.69±0.05,24 h:0.75±0.01,48 h:1.01±0.03,P<0.05),而miR-122水平随TGF-β1处理时间的延长而逐渐下降(0 h:0.72±0.05,12 h:0.45±0.01,24 h:0.37±0.03,48 h:0.29±0.08,P<0.05);与miR-122阴性对照转染组比,miR-122模拟物转染组细胞miR-122水平显著增加(178.45±30.62对12.18±2.39,P<0.01);Western Blot检测发现miR-122模拟物转染组细胞α-SMA蛋白表达水平显著下调;采用TGF-β1处理HST-T6细胞,与miR-122阴性对照转染组比,miR-122模拟物转染组细胞增殖活力显著降低(P<0.05).结论肝纤维化组织细胞miR-122水平下调,而过表达miR-122可抑制肝星状细胞的活化和增殖,从而可能抑制肝纤维化的发生和发展.Objective The purpose of this experiment was to explore the roles of microRNA(miR)-122 in the pathogenesis of liver fibrosis by in vitro and by in vivo.Methods The liver fibrosis model was established in C57BL/6 mice by intraperitoneal injection of carbon tetrachloride and also in HSC-T6 cells in vitro by incubation with 10 ng/ml transforming growth factor-β1(TGF-β1).The miR-122 agomir and miR-122 mimics were transfected to overexpress miR-122 in mice and in hepatic stellate cells.The total RNA and whole protein were extracted for RT-PCR and Western-blot detection of miR-122,α-smooth muscle actin(α-SMA),type I collagen(CollagenⅠ),tissue inhibitor of metalloproteinase 1(TIMP-1),and platelet-derived growth factor(PDGF).The proliferation of HSC-T6 cells was detected by CCK-8.Results The expression ofα-SMA in the liver tissue in the model animal was significantly higher than that in the control group(9.92±2.12 vs.1.12±0.54,P<0.01),while the miR-122 level was significantly lower than that in the control group(0.95±0.31 vs.2.07±0.28,P<0.01);in mice with carbon tetrachloride-induced liver fibrosis,the miR-122 level in the miR-122 agomir-transfected group was significantly higher than that in the miR-122 agomir control-transfected group(6.27±1.73 vs.2.78±0.21,P<0.01);theα-SMA,type I collagen,TIMP-1 and PDGF protein expression in the miR-122 agomir-transfected group were significantly decreased;in TGF-β1-intervened HST-T6 cells,theα-SMA expression increased as the prolongation of TGF-β1 treatment(0h:0.61±0.02,12 h:0.69±0.05,24 h:0.75±0.01,48 h:1.01±0.03,P<0.05),while the miR-122 levels decreased(0 h:0.72±0.05,12 h:0.45±0.01,24 h:0.37±0.03,48 h:0.29±0.08,P<0.05);the miR-122 level in the miR-122 mimics-transfected cells greatly increased as compared with the miR-122 negative control-transfected cells(178.45±30.62 vs.12.18±2.39,P<0.01);the Western blot showed that the expression ofα-SMA protein was significantly down-regulated in the miR-122 mimics-transfected group;in TGF-β1-intervened HST-T6 ce

关 键 词：肝纤维化 HSC-T6细胞 微小RNA 金属蛋白酶组织抑制因子1 血小板衍生生长因子 小鼠 

分 类 号：R575.2[医药卫生—消化系统]