Calpain2及其抑制剂在肝纤维化细胞凋亡中的机制  

Mechanism of calpain 2 and its inhibitor calpastatin in the apoptosis of hepatic cells during liver fibrosis

作　　者：谢汝佳[1] 韩冰[1] 杨婷[1] 邹河[1] 杨勤[1] 

机构地区：[1]贵阳医学院病理生理教研室,贵阳550004

出　　处：《重庆医科大学学报》2014年第7期960-963,共4页Journal of Chongqing Medical University

基　　金：贵州省科技厅国际科技合作资助项目(编号:黔科合外G字【2010】7016号)

摘　　要：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 observe the changes in expressions of calpain2 and its inhibitor calpastatin cluring liver fibrosis and to explore their effects on apoptosis of hepatic cells. Methods：Forty male Wistar rats were randomly divided into four groups（4 weeks normal control group, 8 weeks normal control group,4 weeks liver fibrosis group and 8 weeks liver fibrosis group）,each group in- eluded 10 rats. Groups were induced by hypodermic injection of 40% CCI4 every 3 d for 4 weeks and 8 weeks respectively,the dosage of CCI4 is 0.3 ml/100 g body weight. Apoptosis of hepatic ceils was detected by TUNEL. Additionally,protein expressions of calpain 2,ealpastatin and activated easpase 3 were determined by immunohistochemistry and Western blot and mRNA expressions of cal- pain2 and ealpastatin were determined by real-time PCR. Results：Results of immunohistochemistry and Western blot revealed that there was no difference in expressions of calpain 2 and ealpastatin protein between 4 weeks fibrosis group and normal control group （P〉O.05）. In 8 weeks fibrosis group, expressions of calpain2 were increased obviously and expressions of ealpastatin were decreased with the increase of liver fibrosis degree. Additionally, real-time PCR results revealed that expressions of calpain2 mRNA in 4 weeks and 8 weeks fibrosis groups were elevated compared with those in normal control group（P〈0.01 ）, while expressions of ealpastatin mRNA were decreased in 8 weeks fibrosis group（P〈0.01 ）. Expressions of activated easpase3 protein in 4 weeks and 8 weeks fibrosis groups were increased compared with those in normal control group and the number of apoptotie cells were also elevated in fibrosis groups than in normal control group （P〈0.01）. Conclusion：Calpain2 and ealpastatin may play important roles in the process of liver fibrosis. Their effects on fibrosis might be exerted through promoting the apoptosis of hepatic cells.

关 键 词：肝纤维化 钙中性蛋白酶2 CALPASTATIN 

分 类 号：R363[医药卫生—病理学]