腺相关病毒介导的肝细胞核因子1α过表达改善四氯化碳诱导的小鼠肝纤维化

Overexpression of hepatocyte nuclear factor 1α mediated by adeno-associated virus attenuates carbon tetrachloride-induced hepatic fibrosis in mice

目的探讨特异性上调肝细胞中肝细胞核因子1α(HNF1α)表达对四氯化碳(CCl4)诱导的小鼠肝纤维化的影响。方法取18只C57/B6小鼠随机分为正常组、AAV8-TBG-Ctrl组和AAV8-TBG-HNF1α组,每组6只。应用CCl4诱导制备小鼠肝纤维化模型,造模后AAV8-TBG-HNF1α组小鼠通过尾静脉注射带有甲状腺素结合球蛋白(TBG)启动子驱动表达HNF1α的腺相关病毒AAV8-TBG-HNF1α特异性上调肝细胞中HNF1α的表达,AAV8-TBGCtrl组小鼠注射对照病毒AAV8-TBG。采用免疫组织化学法及qPCR检测各组小鼠HNF1α的表达,苏木素-伊红(H-E)染色、天狼猩红染色检测肝组织的病理改变及胶原沉积,免疫组化法检测α-平滑肌肌动蛋白(α-SMA)的表达,并对Ⅰ型胶原(COL1A1)及α-SMA的表达进行软件定量分析;qPCR法检测小鼠肝组织中纤维化相关基因(α-SMA和COL1A1)、上皮指标(E-cadherin和Plakoglobin)及间质指标(Vimentin,Slug和Twist1)的mRNA水平;免疫组化法及TUNEL法检测上调HNF1α对纤维化肝脏中细胞增殖、凋亡的影响。结果与正常组小鼠相比,CCl4造模可促进小鼠肝脏胶原沉积及α-SMA的表达,且在肝纤维化发生过程中HNF1α的表达下降(P<0.01);与AAV8-TBG-Ctrl组相比,应用AAV8-TBG-HNF1α特异性上调肝细胞HNF1α的表达可抑制纤维化肝脏中的COL1A1及α-SMA的水平(P<0.01)。上调肝细胞HNF1α表达对纤维化肝脏中E-cadherin、Vimentin等上皮间质转换指标的表达无明显影响(P>0.05),也不影响肝细胞的增殖与凋亡(P>0.05)。结论特异性上调肝细胞HNF1α表达可显著改善CCl4诱导的小鼠肝纤维化。

To explore the influence of the hepatocyte-specific up-regulation of hepatocyte nuclear factor 1α（HNF1α） on mouse hepatic fibrosis induced by carbon tetrachloride（CCl4）.Methods Eighteen C57/B6 male mice were randomly divided into normal group,AAV8-TBG-Ctrl group and AAV8-TBG-HNF1αgroup,with 6mice in each group.Mice in the AAV8-TBG-Ctrl and AAV8-TBG-HNF1αgroups were intraperitoneally injected with CCl4 to establish the hepatic fibrosis mouse model,and then the mice in the AAV8-TBG-HNF1αgroup were injected with AAV8-TBGHNF1αcarrying HNF1αgene under the control of the thyroid-binding globulin（TBG）promoter to specifically upregulate expression of HNF1αin hepatocytes,while the mice in the AAV8-TBG-Ctrl group were injected with control vector AAV8-TBG.The expression of HNF1αwas determined by immunohistochemistry and qPCR.The pathological changes and collagen deposition of liver tissues were detected by hematoxylin-eosin（H-E）staining and sirius red staining,respectively.Immunohistochemistry method was used to detectα-smooth muscle actin（α-SMA）,and the expression of fibrosis related genes（typeⅠcollagenα1chain[COL1A1],α-SMA）,epithelial related genes（E-cadherin,Plakoglobin）and mesenchymal related genes（Vimentin,Slug and Twist1）in liver tissues were analyzed by qPCR.The cell proliferation and apoptosis in fibrotic livers were detected by immunohistochemistry and TdT-mediated dUTP NickEnd Labeling（TUNEL）method,respectively.Results Compared with the normal mice,CCl4 promoted collagen deposition and the expression ofα-SMA in livers,and the expression of HNF1α was significantly decreased（P〈0.01）in the process of hepatic fibrosis.Compared with the AAV8-TBG-Ctrl group,the expression of COL1A1 and α-SMA in the fibrotic livers in the AAV8-TBG-HNF1αg roup was significantly decreased（P〈0.01）.There was no significant difference in the expression of E-cadherin,Vimentin or other Epithelial-mesenchymal transition related genes,or in the cell proliferation and apoptosis between the AAV8-TBG-Ctrl and AAV8-TBG-HNF1α groups（P〉0.05）.Conclusion Hepatocyte-specific up-regulation of HNF1αsignificantly improves CCl4-induced liver fibrosis in mice.