慢性肝病常用动物模型及建模方案

Establishment of animal models of chronic liver disease and modeling methods

目的构建稳定可重复的慢性肝损伤动物模型。方法通过改良高脂饲料、四氯化碳(CCL 4)、硫代乙酰胺(thioacetamide,TAA)分别建立小鼠非酒精性脂肪性肝炎模型(non-alcoholic steatohepatitis,NASH)、小鼠肝纤维化模型、大鼠肝硬化模型。苏木精-伊红(hematoxylin eosin,HE)、油红O、天狼星红(sirius red,SR)染色观察动物肝组织结构变化、脂肪样变以及纤维化程度。自动生化仪检测血清丙氨酸氨基转移酶(alanine transaminase,ALT)、天门冬氨酸氨基转移酶(aspartate aminotransferase,AST)、乳酸脱氢酶(lactate dehydrogenase,LDH)、γ-谷氨酰转移酶(γ-glutamyltransferase,GGT)、甘氨酰脯氨酸二肽氨基肽酶(glycyl proline dipeptidyl aminopeptidase,GPDA)、白蛋白(albumin,Alb)、总胆固醇(total cholesterol,TC)、三酰甘油(triglyceride,TG)。结果改良高脂喂养(64只)与普通饲料喂养的小鼠(8只)相比,ALT为(268.7±69.8)比(35.0±21.9)U/L、AST为(215.0±91.0)比(34.4±9.4)U/L、LDH为(560.3±158.5)比(240.6±101.3)U/L,均明显升高(P<0.05),TC为(1.5±0.3)比(2.2±0.2)mmol/L、TG为(0.9±0.1)比(1.6±0.2)mmol/L,均明显降低(P<0.05)。病理结果显示,改良高脂喂养的小鼠其肝组织内有大量脂滴。注射10%CCL 4(52只)与注射橄榄油(8只)的小鼠相比,ALT为(8507.3±1083.1)比(41.8±29.2)U/L、AST为(4911.2±644.0)比(104.0±33.6)U/L,均明显升高(P<0.05),病理结果显示,注射CCL 4的小鼠其肝组织内有大量纤维化胶原增生。注射TAA(37只)与注射0.9%NaCl(8只)的大鼠相比,ALT为(197.3±131.1)比(34.0±6.0)U/L、AST为(590.3±457.7)比(57.5±12.3)U/L、GGT为(10.0±8.4)比(3.6±3.3)U/L、GPDA为(290.5±134.4)比(63.3±14.7)U/L,均明显升高(P<0.05),Alb为(37.3±1.9)比(40.9±1.3)g/L,明显降低(P<0.05),病理学结果显示,注射TAA的大鼠肝组织内纤维化增生且连接成大小不等的假小叶。结论采用改良高脂连续喂养2周,小鼠NASH成模率可达100%,继续喂养至4周,进一步加重。采用10%CCL 4注射12周,小鼠肝纤维化成模率可达100%,停药后模型至少可维持2周。采用TAA注射16周,大鼠肝硬化成模率可达100%,停药后模型至少可维持4周。

Objective To establish an ideal model of chronic liver injury and evaluate stability of the modeling methods.Methods In this study,the mouse model of non-alcoholic steatohepatitis(NASH),the mouse model of liver fibrosis and the rat model of cirrhosis were induced by the modified high-fat diet(HFD),carbon tetrachloride(CCl4)and thioacetamide(TAA),respectively.Pathological hematoxylin-eosin(HE),oil red O and sirius red(SR)staining were used to observe the structural changes,steatosis and fibrosis of animal liver.Serum alanine transaminase(ALT),aspartate aminotransferase(AST),lactate dehydrogenase(LDH),γ-glutamyltransferase(GGT),glycyl proline dipeptidyl aminopeptidase(GPDA),albumin(ALB),total cholesterol(TC)and triglyceride(TG)were detected by automatic biochemical instrument.Results Compared with traditional diet group(8 mice),the modified HFD group(64 mice)could increase the activities of ALT[(268.7±69.8)vs(35.0±21.9)],AST[(215.0±91.0)vs(34.4±9.4)]and LDH[(560.3±158.5)vs(240.6±101.3)]significantly(all P<0.05),and decrease the activities of TC[(1.5±0.3)vs(2.2±0.2)]and TG[(0.9±0.1)vs(1.6±0.2)]significantly(both P<0.05).In the pathological results,oil red O and HE staining were observed in pathology showed that the hepatic tissue of mice fed with the modified HFD were filled with lipid droplets.Compared with mice injected with olive oil(8 mice),levels of ALT[(8507.3±1083.1)vs(41.8±29.2)]and AST[(4911.2±644.0)vs(104.0±33.6)]increased significantly(P<0.05)in mice injected with 10%CCl4(2mL/kg,tiw)for 12 weeks(52 mice).The pathological results of HE and SR staining showed that there was a large number of collagen fibers in the liver tissue of mice injected with CCl4.Compared with mice injected with normal saline(8 rats),levels of ALT[(197.3±131.1)vs(34.0±6.0)],AST[(590.3±457.7)vs(57.5±12.3)],GGT[(10.0±8.4)vs(3.6±3.3)]and GPDA[(290.5±134.4)vs(63.3±14.7)]increased significantly(all P<0.05)and level of ALB[(37.3±1.9)vs(40.9±1.3)]decreased significantly(P<0.05)in rat injected with TAA(200mg/kg,biw)for 16 weeks(37 rats).The pathological analysis of HE and SR staining showed that the liver tissue of rats injected with TAA presented with hepatic fibrosis,proliferation of hepatic fibroblasts and formed pseudo lobules with different sizes.Conclusion Mice were fed with the modified HFD for 2 weeks,the success rate of NASH model was 100%.The model was further aggravated after feeding for 4 weeks.In terms of mice that were injected with 10%CCl4(2 mL/kg,tiw)for 12 weeks,the success rate of liver fibrosis model was 100%,and the model could be maintained for at least 2 weeks after drug withdrawal.Rats were injected with TAA(200mg/kg,biw)for 16 weeks,the success rate of liver cirrhosis model was 100%,and the model could last for at least 4 weeks after drug withdrawal.