Vinculin促小鼠肝星型细胞活化作用

Vinculin promotes hepatic stellate cells activation in mice

目的研究Vinculin在小鼠肝纤维化模型中的表达及作用。方法成年雄性昆明小鼠25只,随机分为对照组(n=10)和实验组(n=15)。实验组皮下注射四氯化碳-粟米油混合液(6 mL/kg),2次/周,对照组皮下注射同等剂量的粟米油。造模8周后取小鼠中叶肝组织行HE染色及Masson染色检测小鼠肝脏结构变化,免疫组织化学检测Desmin,Vinculin在肝内的表达及细胞定位。人源性肝星形细胞系LX-2瞬时转染Vinculin siRNA,qPCR及蛋白质印迹检测敲减Vinculin对desmine、α-SMA的表达的影响,ELISA检测敲减Vinculin后Ⅰ型胶原纤维的生成变化。结果 CCl4处理8周后,小鼠肝脏汇管区及肝小叶周围胶原纤维增多,局部有假小叶生成趋势,免疫组化结果显示与对照组相比desmin及Vinculin阳性细胞数量增多(差异有统计学意义,P<0.05),Vinculin特异性表达于活化肝星形细胞胞膜上;qPCR结果显示敲减星形细胞Vinculin表达后,desmine和α-SMA表达量下降(差异有统计学意义,P<0.05),Ⅰ型胶原纤维表达量无明显下降。结论 Vinculin介导肝星形细胞的活化。

Objective To investigate the expression and effect of Vinculin in the mouse model of liver fibrosis.Methods Twenty-five adult male mice were randomly divided into a control group(n=10)and an experimental group(n=15).The experimental group was subcutaneously injected with carbon tetrachloride(CCl4)-corn oil mixture(6 ml/kg mass)2 times/week,and the control group was subcutaneously injected with corn oil at the same dose.After 8 weeks of modeling,the changes of liver structure were observed through the tissue of middle liver lobe using HE staining and Masson staining.The expression and cell localization of desmine and Vinculin in the liver were detected by immunohistochemistry.Vinculin were knocked down in human hepatic stellate cell line LX-2 by transient transfection of small interfering RNA.Quantitative real-time polymerase chain reaction(qPCR)and Western Blot was performed to investigate the impact of knocking down Vinculin on desmin and alpha SMA expression.The content of typeⅠ collagen fiber after knocking down Vinculin was detected by enzyme-linked immunosorbent assay.Results After 8-week CCl4 treatment,collagen fibers increased in the portal and perilobular areas,and pseudolobule formation was observed.The immunohistochemistry showed that the number of desmin and Vinculin positive cells increased in the experimental group compared with the control group with significant differences(P<0.05),and that Vinculin was specifically expressed on the membrane of activated stellate cells.The qPCR showed that the expression of desmine and alpha SMA decreased after knocking out Vinculin with significant differences(P<0.05),and that the content of type I collagen fiber didn’t decrease significantly.Conclusion Vinculin mediates the activation of hepatic stellate cells.