长爪沙鼠NAFLD模型中CIP4基因的表达及与β-catenin蛋白相互作用的免疫共沉淀验证  

Expression of CIP4 gene and its interaction with β-catenin protein in gerbil nonalcoholic fatty liver disease model

作　　者：吴鸿儒 吴旧生[2] 王志远[3] 刘月环[2] WU Hongru;WU Jiusheng;WANG Zhiyuan(Department of Gastroenterology,Zhejiang Greentown Cardiovascular Hospital,Hangzhou 310012,China)

机构地区：[1]浙江绿城心血管病医院消化内科,杭州310012 [2]浙江大学动物科学学院 [3]浙江省医学科学院 [4]江大学医学院

出　　处：《浙江医学》2020年第10期995-999,I0003,共6页Zhejiang Medical Journal

基　　金：浙江省自然科学基金项目(LY16H030011);浙江省科技厅公益技术应用研究计划项目(2011C37096,2015C37102);浙江省医药卫生平台骨干人才基金项目(2015RCA006)。

摘　　要：目的获得长爪沙鼠非酒精性脂肪性肝病(NAFLD)模型中CDC42作用蛋白4(CIP4)基因的表达规律及是否受β连环蛋白(β-catenin)调控的证据。方法3月龄雄性长爪沙鼠30只采用随机数字表法分为正常组6只和模型组24只,模型组按饲喂高脂饲料的时间不同分为2周组、4周组、8周组、12周组,每组6只。按组采集肝脏大叶标本4份,HE染色及Masson染色检测肝脏的病理变化,荧光定量PCR(qPCR)法检测CIP4 mRNA相对表达量,蛋白免疫印迹(Western blot)法检测CIP4蛋白相对表达量,免疫共沉淀(CoIP)验证CIP4与β-catenin的相互作用。结果肝病理学检测结果显示,随着造模时间的延长,模型组沙鼠均出现肝脏脂肪病变(单纯脂肪肝,2~4周造模,S0~S1),脂肪性肝炎(4~8周造模,脂堆积形成肝细胞的肿胀,坏死和空泡,少量纤维化出现,S1~S2),肝纤维化(8~12周造模,S2~S3),部分有假小叶形成。qPCR和Western blot结果显示,随着造模时间延长,CIP4 mRNA和蛋白相对表达量均下降。模型组被CoIP下来的CIP4蛋白明显下降,这表明模型组β-catenin与CIP4的相互作用减少。结论随着NAFLD病程的进展,长爪沙鼠模型CIP4呈现降低趋势,其作用受β-catenin的调控。Objective To investigate the expression of CIP4 gene and its interaction withβ-catenin in Mongolian gerbils with nonalcoholic fatty liver disease(NAFLD).Methods Thirty male young gerbils were divided into control group(n=6)and NAFLD model group(n=24);the model group were further divided into 2M,4M,8M and 12M groups with 6 in each subgroup according to the time of feeding with high fat diets.The liver specimens were taken for pathological observation with HE staining,Masson staining,and the expression of the CIP4 mRNA and protein in liver tissue was determined by qPCR and Western blot,the interaction of CIP4 withβ-catenin was examined by co-immunoprecipitation(CoIP).Results The hepatic fibrosis were developed within 8-12 weeks,the liver sections presented blue-stained collagen fibers as the features of a S2-S3 fibrosis phase.qPCR results showed that the transcriptional level of CIP4 gene gradually decreasing with the modeling time.Western blot results showed that the expression level of CIP4 gene also reduced with the development of liver fibrosis.The results of CoIP exhibited that the content of CIP4 protein was significantly decreased by the same amount ofβ-catenin protein IP.Conclusion CIP4 shows a low expression trend with the extension of modeling time of NAFLD in gerbils,which may be regulated byβ-catenin.

关 键 词：非酒精性脂肪性肝病模型 长爪沙鼠 CDC42作用蛋白基因 免疫共沉淀 

分 类 号：R575.5[医药卫生—消化系统] R-332[医药卫生—内科学]