四氯化碳诱导C57BL/6J小鼠肝纤维化模型建立方法及优化

Establishment and optimization of C57BL/6J mouse liver fibrosis model induced by carbon tetrachloride

目的通过不同给药方式不同剂量四氯化碳诱导的C57BL/6J小鼠肝纤维化模型,通过影像学、分子生物学及组织病理学等方法进行比较,以此优化四氯化碳诱导的小鼠肝纤维化模型。方法36只健康C57BL/6J雄性小鼠,适应性饲养1周后,随机分为空白组、2周组、3周组、4周组、6周组及8周组共6组(n=6),除空白组外均作为阳性对照组。空白组以橄榄油0.2 mL腹腔注射,每周3次,各阳性对照组以20%CCl4-橄榄油溶液腹腔注射,剂量为2 mL/kg,每周3次。记录各组小鼠体重变化情况。分别于第15、22、29、43及57天测量小鼠肝弹性值后取血,分别测量小鼠谷丙转氨酶(alanine aminotransferase,ALT)、天门冬氨酸氨基转移酶(aspartate aminotransferase,AST)、透明质酸(hyaluronic acid,HA)、层黏蛋白(laminin,LN)、前Ⅲ型前胶原(pro-typeⅢcollagen,PC-Ⅲ)及Ⅳ型胶原(typeⅣcollagen,Ⅳ-C)含量,对肝组织进行苏木精-伊红(hematoxylin and eosin,HE)、Masson及天狼星红染色,Metavir评分系统评估肝纤维化程度。结果与空白组相比,各阳性对照组小鼠精神萎靡、扎堆喜卧。在小鼠体重方面,4周组、6周组、8周组与空白组相比均明显下降,而2周组与空白组相比体重明显升高。肝弹性超声显示,随着给药时间增加,弹性值呈进行性升高。生化结果显示,与空白组相比,各阳性对照组ALT、AST水平均显著升高,随着给药时间增加,各阳性对照组HA、LN、PC-Ⅲ及Ⅳ-C水平呈升高趋势。病理结果显示:随着给药时间增加,小鼠肝纤维化程度呈进行性加重,4周时符合肝纤维化病理诊断,6周时有假小叶形成趋势,而8周时已形成假小叶,提示早期肝硬化。结论20%CCl4-橄榄油溶液以每周3次、连续4周腹腔注射的方法能成功建立C57BL/6J小鼠肝纤维化模型,稳定性好、成模较快,可以作为肝纤维化模型制造的优化方案。

Objective To optimize a C57BL/6J mouse liver fibrosis model induced by different doses of carbon tetrachloride through imaging,molecular biology,and pathology method.Methods Thirty⁃six healthy C57BL/6J male mice were randomly divided into a control group,2 weeks,3 weeks,4 weeks,6 weeks,and 8 weeks groups(n=6)after adaptive feeding for 1 week.The control group was intraperitoneally injected with 0.2 mL olive oil three times a week,and the positive⁃control groups were intraperitoneally injected with 0.2 mL 20%CCl4⁃olive oil solution three times a week.Changes in the body weights of mice in each group were recorded.Liver stiffness was measured on days 15,22,29,43 and 57,and blood samples were collected,and cereal third alanine aminotransferase(ALT),aspartate aminotransferase(AST),hyaluronic acid(HA),laminin(LN),pro⁃typeⅢcollagen(PC⁃Ⅲ),and typeⅣcollagen(Ⅳ⁃C)content was measured.The liver tissues were stained with hematoxylin and eosin(HE),Masson,and Sirius red.The Metavir scoring system was used to evaluate the degree of liver fibrosis.Results Compared with the control group,mice in the positive⁃control groups were listless and tended to huddle together.In terms of body weight,the 4 weeks,6 weeks,and 8 weeks groups were significantly lighter than the control group,while the 2 weeks group mice were significantly heavier than the control group mice.Liver elastography showed a progressive increase in stiffness with increased administration time.The biochemical tests showed that,compared with the control group,the other groups’ALT and AST levels were significantly higher.With an increase in drug delivery time,the positive⁃control group’s HA,LN,PC⁃ⅢandⅣ⁃C levels showed increasing trends.Pathological examination revealed that liver fibrosis was progressively aggravated with an increase in administration time.At 4 weeks,the pathological diagnosis was consistent with that of liver fibrosis,and there were signs of pseudolobule formation at 6 weeks.Pseudolobules were formed at 8 weeks,suggesting early cirrhosis.Conclusions A liver fibrosis model can be successfully established in C57BL/6J mice by intraperitoneal injection of 20%CCl4⁃olive oil solution three times a week for 4 consecutive weeks.The model has good stability,and the modeling method is rapid and can be used as an optimized scheme for the establishment of liver fibrosis models.