细胞自噬在D-氨基半乳糖／脂多糖诱导小鼠急性肝衰竭中的表达  

Role of autophagy in acute liver failure induced by D - galactosamine/lipopolysaccharide in mice

作　　者：魏琳琳[1] 张向颖[1] 张莉[1] 杨蓉蓉[1] 时红波[1] 温韬[1] 陈德喜[1] 段钟平[1] 任锋[1] 

机构地区：[1]首都医科大学附属北京佑安医院,北京100069

出　　处：《中华肝脏病杂志》2016年第8期608-613,共6页Chinese Journal of Hepatology

基　　金：国家十二五科技重大专项（2012zx100020004-006,2012zx10004904-003-001,2013ZX10002002-006-001）;国家自然科学基金项目（81270532）;北京市自然科学基金（7162085）;首都特色临床应用研究（Z121107001012167）;王宝恩肝纤维化研究基金项目（CFHPC20131031）;北京市卫生系统高层次卫生技术人才医学骨干培养资助项目（2013-3-075）

摘　　要：目的利用D-氨基半乳糖／脂多糖（D—GalN／LPS）诱导的小鼠急性肝衰竭模型，研究细胞自噬在肝衰竭疾病进展过程中的表达及其作用。方法以C57BL／6小鼠为研究对象，腹腔注射nGalN／uIS建立小鼠急性肝衰竭模型。动物实验分组：对照组、D—GalN／LPS诱导肝衰竭模型2h组、D-GalN／LPS诱导肝衰竭模型4h组和D-GalN／LPS诱导肝衰竭模型6h组。检测血清丙氨酸氨基转移酶（ALT）、天冬氨酸氨基转移酶（AST）评价肝脏功能，观察肝脏组织病理变化评价肝脏损伤情况，实时荧光定量PCR检测自噬相关基因表达，Westernblot测定小鼠肝组织中自噬相关蛋白表达，荧光显微镜观察肝衰竭进展过程中自噬体的表达情况。多组样本均数的两两比较采用One-wayANOVA分析（方差齐者用LSD—t检验，方差不齐者用Games-Howell法），尸〈0．05为差异有统计学意义。结果急性肝衰竭模型组肝功能随着D—GalN／I鹧刺激的进展逐渐受损：ALT、AST在4h后显著增高，肝脏组织病理损伤随时间逐渐加重，6h达到急性肝衰竭标准；自噬相关基因ATG-7、ATG-5、Beclin-1、Lamp-1和LC3amRNA和蛋白在急性肝损伤早中期（2h和4h）表达增高，而进展至肝衰竭（6h）后表达下降；荧光显微镜观察发现给予D—GalN／LPS在4h时间点自噬体的表达最多。结论在D-氨基半乳糖／脂多糖诱导急性肝损伤早中期，自噬表达逐渐增强，而进展至肝衰竭后自噬表达下降，因此细胞自噬可能在急性肝衰竭的发病机制中发挥着重要作用。Objective To investigate the expression and role of autophagy in the progression of acute liver failure （ALF） using the mouse model of ALF induced by D-galactosamine/LPS （D-GalN/LPS）. Methods The C57BL/6 mice were used, and intraperitoneal injection of D-galactosamine （D-GaIN） and lipopolysaccharide （LPS） was performed to establish the mouse model of ALE The mice were divided into control group and 2-, 4-, and 6-hour D-GalN/LPS-induced ALF model groups. The serum levels of alanine aminotransferase （ALT） and aspartate aminotransferase （AST） were measured to assess liver function, and the pathological changes in liver tissue were observed to evaluate the status of liver injury. Quantitative real-time PCR was used to measure the expression of autophagy-related genes, Westem blot was used to measure the expression of autophagy-related proteins in liver tissue, and a fluorescence microscope was used to observe the expression of autophagosome in the progression of liver failure. A one-way ANOVA was used for comparison of means of multiple samples between any two groups （LSD-t test for data with homogeneity of variance and Games-Howell method for data with heterogeneity of variance）. P 〈 0.05 was considered statistically significant. Results The ALF model groups showed gradual liver impairment over the time of D-GalN/LPS stimulation. There were significant increases in ALT and AST after 4 hours; the pathological injury of liver tissue gradually aggravated over the time of D-GalN/LPS stimulation and fulfilled the criteria for ALF at 6 hours. The mRNA and protein expression of autophagy-related genes （ATG-7, ATG-5, Beclin-1, Lamp-l, and LC3a） increased in the early and medium stages of ALF （2 and 4 hours） and decreased after ALF progressed to liver failure （6 hours）. As was observed via the fluorescence microscope, the 4-hour D-GalN/LPS-induced ALF model group showed the highest expression of autophagosome. Conclusion The expression of autophagy gradually increases in the early an

关 键 词：自噬 肝脏 脂多糖类 肝功能衰竭 急眭 

分 类 号：R575.3[医药卫生—消化系统]