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目的探讨肝纤维化发生过程中β-连环蛋白(β-catenin)定位、表达及意义。方法健康雄性昆明小鼠(n=45)随机分为对照组(n=15)与实验组(n=30)。实验组小鼠皮下注射50%四氯化碳(CCl4)-粟米油混合液(6ml/kg),2次∕周,对照组皮下注射同等剂量的粟米油。各组分别于造模1周、4周和8周后取小鼠肝脏,常规制作石蜡切片,Masson染色及Desmin免疫组织化学法观察比较不同组别小鼠肝纤维化病理变化,RT-PCR及免疫组织化学方法检测不同时间点各组小鼠肝组织内β-catenin的表达及定位。结果 Masson染色结果显示,对照组肝汇管区结缔组织内及血管壁有少量细小纤维,实验组小鼠肝脏汇管区和中央静脉及其周围胶原纤维增多;随损伤时间延长纤维增生愈加明显。Desmin免疫组织化学结果显示,各组别均有阳性表达,但损伤组各时间点desmin阳性表达细胞数明显高于对照组(P<0.05或P<0.001)。RT-PCR结果显示,CCl4损伤1周后肝内β-cateninmRNA水平与对照组相比无明显差别(P>0.05),损伤4周及8周β-catenin mRNA水平则明显下降,与对照组相比差异均有显著性(P<0.01)。免疫组织化学结果显示,对照组β-catenin弱表达于肝细胞膜及胆管上皮细胞膜和胞质,而损伤组β-catenin阳性反应主要定位于肝内增生的细胞团及新生胆管上皮细胞质,各组间积分吸光度值差别有显著性(P<0.01)。结论 CCl4诱导的肝纤维化过程中β-catenin mRNA表达与蛋白表达不同步,阳性表达细胞主要为新生的细胞团及胆管上皮细胞。
Objective To investigate the localization, expression and significance of β-catenin in the pathogenesis of hepatic fibrosis. Methods Healthy male Kunming mice (n = 45) were randomly divided into control group (n = 15) and experimental group (n = 30). The mice in the experimental group were subcutaneously injected with CCl4-corn oil (6ml / kg) twice a week, and the control group was injected subcutaneously with the same amount of corn oil. The mice were sacrificed at 1 week, 4 weeks and 8 weeks after model establishment. Paraffin sections were made by routine paraffin sections. Masson staining and Desmin immunohistochemistry were used to observe the pathological changes of liver fibrosis in different groups. RT-PCR and Immunohistochemistry was used to detect the expression and localization of β-catenin in liver tissues of mice in different time points. Results The results of Masson staining showed that a small amount of fine fibers were found in the connective tissue and in the vascular wall of the liver portal area in the control group. Collagen fibers in the portal area and the central vein of the experimental group and the surrounding vein were increased in the control group. Fibrosis was more obvious with prolonged injury time. Desmin immunohistochemistry results showed that each group had positive expression, but the number of desmin positive cells at each time point in the injury group was significantly higher than that in the control group (P <0.05 or P <0.001). The results of RT-PCR showed that there was no significant difference in the level of β-catenin mRNA between the CCl4 group and the control group (P> 0.05), and the level of β-catenin mRNA decreased significantly at 4 and 8 weeks The differences were significant (P <0.01). The results of immunohistochemistry showed that the β-catenin in the control group was weakly expressed in the membrane and the cytoplasm of the hepatocyte membrane and the bile duct epithelial cells, whereas the β-catenin positive reaction in the lesion group mainly localized in the intrahepatic proliferative cell mass and the nascent biliary epithelial cytoplasm. The difference of integral absorbance was significant (P <0.01). Conclusion The expression of β-catenin mRNA and protein in CCl4-induced hepatic fibrosis are not synchronized, and the positive cells are mainly nascent cell mass and bile duct epithelial cells.