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目的:探讨钙中性蛋白酶2(calpain2)及其抑制剂calpastatin在肝纤维化过程中的表达变化及他们在肝细胞凋亡中的作用。方法:雄性Wistar大鼠40只,随机分为正常组4、8周组和肝纤维化4、8周组,每组各10只。肝纤维化组按0.3 ml/100 g体质量皮下注射40%CCl4植物油溶液,每隔3 d注射1次,造模时间分别为4周和8周;正常组大鼠皮下注射等量植物油溶液。采用原位末端转移酶标记(terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling,TUNEL)法检测肝组织中肝细胞凋亡的情况;免疫组织化学法及Western blot检测肝组织中calpain2、calpastatin及活性caspase3的表达情况;realtime PCR检测肝组织中calpain2及calpastatin mRNA的表达变化。结果:免疫组化及Western blot检测显示肝纤维化4周组大鼠肝组织中calpain2及calpastatin蛋白的表达与正常4周组比较差异无统计学意义(P>0.05);随着肝纤维化程度的加重,肝纤维化8周时大鼠肝组织中calpain2的表达明显增加,而calpastatin的表达明显减少;real-time PCR检测发现肝纤维化4、8周组大鼠肝组织中calpain2 mRNA表达较相应的正常对照组明显增加(P<0.01),而calpastatin mRNA的表达在肝纤维化8周组明显减少(P<0.01);此外,通过免疫组化及Western blot发现肝组织中活性caspase3的表达在肝纤维化4周时已明显增加,肝纤维化8周时达高峰;同时TUNEL法检测发现肝纤维化大鼠肝组织中肝细胞凋亡的数目较正常组大鼠显著增加(P<0.01)。结论:calpain2与calpastatin在蛋白及基因水平的表达变化,提示他们可能参与了肝纤维化的发生发展过程,其致肝纤维化的机制可能与促进肝细胞的凋亡有关。
Objective: To investigate the changes of calpain2 and its inhibitor calpastatin during hepatic fibrosis and their roles in hepatocyte apoptosis. Methods: Forty male Wistar rats were randomly divided into normal 4 weeks, 8 weeks and 4 weeks and 8 weeks of hepatic fibrosis, 10 rats in each group. The liver fibrosis group was subcutaneously injected with 40% CCl4 vegetable oil solution at a volume of 0.3 ml / 100 g once every 3 days for 4 weeks and 8 weeks respectively. The rats in the normal group were injected subcutaneously with the same amount of vegetable oil solution. Liver cell apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL). The expressions of calpain2, calpastatin and activity in liver tissue were detected by immunohistochemistry and Western blot caspase3 expression; realtime PCR detection of liver tissue calpain2 and calpastatin mRNA expression changes. Results: The expression of calpain2 and calpastatin protein in the liver tissue of the 4-week liver fibrosis group showed no significant difference compared with the normal 4-week group (P> 0.05), but with the degree of liver fibrosis , The expression of calpain2 increased obviously and the expression of calpastatin decreased significantly in liver fibrosis rats after 8 weeks of liver fibrosis. The expression of calpain2 mRNA in liver tissue of 4 and 8 weeks hepatic fibrosis rats after real-time PCR analysis (P <0.01), while the expression of calpastatin mRNA in hepatic fibrosis group was significantly decreased (P <0.01). In addition, the expression of active caspase 3 in liver tissue was detected by immunohistochemistry and Western blot The number of hepatic cells in hepatic fibrosis rats increased significantly at 4 weeks after hepatic fibrosis and peaked at 8 weeks after hepatic fibrosis. The number of hepatic cells in liver fibrosis rats increased significantly (P <0.01) by TUNEL assay. Conclusion: The expression changes of calpain2 and calpastatin at the protein and gene level suggest that they may be involved in the development and progression of hepatic fibrosis. The mechanism of hepatic fibrosis may be related to the promotion of apoptosis in hepatocytes.