四氯化碳诱导兔肝纤维化模型的改良-东南大学学报医学版

四氯化碳诱导兔肝纤维化模型的改良

单位：1. 东南大学医学院, 江苏南京 210009;
2. 东南大学附属中大医院普外科, 江苏南京 210009;
3. 东南大学附属中大医院病理科, 江苏南京 210009

分类号：R575.2;R-332

出版年·卷·期（页码）：2015·34·第四期（562-567）

摘要：

目的:通过改良传统四氯化碳(CCl₄)法诱导兔肝纤维化模型,提高以肝纤维化模型为基础的相关研究的实验效率及动物模型质量。方法:选取50只健康雄性新西兰大白兔,每批25只。首批实验(传统组)将动物随机分为两组,造模组21只,皮下注射50% CCl₄橄榄油溶液,每周两次;对照组4只,相同方法皮下注射纯橄榄油,注射12周,观察记录兔一般情况。造模组于0、4、8、12周行影像学及血清学生化指标检测,每次采血后随机处死7只兔(兔意外死亡时按剩余数量均分计),测门静脉压(PVP)并取组织行HE、Masson、免疫组化染色及ISHAK评分;对照组12周后处死大白兔,其余处理同造模组。针对第1批实验中暴露的问题进行改良,将改良方案应用于第2批(改良组)实验。结果:传统造模组及改良造模组动物死亡率分别为42.9%和9.5%,造模成功率分别为57.1%和85.7%,两组比较差异均有统计学意义(P<0.05)。改良组兔体重、丙氨酸氨基转移酶、天冬氨酸转氨酶、白蛋白、总胆红素与对照组相比随实验进行出现差异有统计学意义的变化(P<0.05);造模组12周时门静脉血流速度较对照组减慢,螺旋CT扫描显示造模组肝实质密度较对照组降低,HE、Masson及免疫组化染色可见肝实质发生明显纤维化改变并可见假小叶形成,造模组4、8、12周时ISHAK评分分别为(1.33±0.52)、(1.83±0.75)、(4.17±0.75)分。结论:通过对CCl₄诱导兔肝纤维化模型的传统造模方案进行针对性改良,可在保证造模成功率的基础上降低动物死亡率,为以动物肝纤维化模型为基础的实验研究打下良好基础。

Objective: To increase experimental efficiency and animal model's quality of fundamental research which based on liver fibrosis model of animal by modify the traditional process of carbon tetrachloride(CCl₄) induced liver fibrosis of rabbit. Methods: 50 healthy male new Zealand rabbits were included in this research and divided into 2 batches on average. Rabbits in first batch(traditional batch) were separated into 2 groups: modeling group(n=21) and control group(n=4). Rabbits in modeling group were injected with 50% CCl₄ olive oil solution subcutaneously twice a week for 12 weeks in succession while control group were treated with the same amount of pure olive oil. Rabbits in modeling group received color Doppler ultrasound, spiral computed tomography and serum biochemical examination every 4 weeks from the beginning to 12 weeks, 7 rabbits(remanent were divided equally when rabbits died unexpected ) were sacrificed after blood drawing every 4 weeks to measure portal venous pressure(PVP) and obtained liver tissues for biopsy and immunohistochemical(IHC) staining as well as staging according the ISHAK grading system; Control group were treated with the same way with modeling group and sacrificed at 12 weeks. Modify disadvantages exposed during experiment of first batch and apply revised scheme to the second batch(modified batch). Results: Mortality of traditional patch was 42.9% and significantly higher than modified batch of 9.5%(P<0.05) and the success rate of traditional patch(57.1%) was obviously lower than that of modified batch(85.7%),too. With progression of modeling process,ALT, AST, ALB, TP of modeling group underwent significant changes compare to control group(P<0.05); color doppler flow imaging showed the speed of portal venous blood flow of modeling group was obviously slower and CT values were significantly reduced comparing to control group; HE, Masson and IHC staining showed explicit liver fibrosis and pseudolobule could be seen, ISHAK score rised over time(1.33±0.52,1.83±0.75,4.17±0.75 of modified modeling group at 4,8,12 week respectively). Conclusion: Continuous injection of CCl4 can induce explicit liver fibrosis of rabbit. By adjust modeling process of modeling process we can reduce mortality of animals drastically and guarantee the success rate at the same time and provide useful information for research based on liver fibrosis animal models.

参考文献：

[1] ELLIS E L,MANN D A.Clinical evidence for the regression of liver fibrosis [J].Journal of Hepatology,2012,56(5):1171-1180.
[2] CASADO J L,QUEREDA C,MORENO A,et al.Regression of liver fibrosis is progressive after sustained virological response to HCV therapy in patients with hepatitis C and HIV coinfection [J].Journal of Viral Hepatitis,2013,20(12):829-837.
[3] KONG D,ZHANG F,ZHANG Z,et al.Clearance of activated stellate cells for hepatic fibrosis regression:molecular basis and translational potential [J].Biomedicine & Pharmacotherapy,2013,67(3):246-250.
[4] DING K,LIU M R,LI J,et al.Establishment of a liver fibrosis model in cynomolgus monkeys [J].Experimental and Toxicologic Pathology,2014,66(5-6):257-261.
[5] WU J,NORTON P A.Animal models of liver fibrosis[J].Scand J Gastroenterol,1996,31(12):1137-1143.
[6] JIANG Y,KANG Y J.Metallothionein gene therapy for chemical-induced liver fibrosis in mice[J].Molecular Therapy:the Journal of the American Society of Gene Therapy,2004,10(6):1130-1139.
[7] JIANG Y,LIU J,WAALKES M,et al.Changes in the gene expression associated with carbon tetrachloride-induced liver fibrosis persist after cessation of dosing in mice[J].Toxicological Sciences,2004,79(2):404-410.
[8] FORT J,PILETTE C,VEAL N,et al.Effects of long-term administration of interferon alpha in two models of liver fibrosis in rats [J].Journal of Hepatology,1998,29(2):263-270.
[9] AHMED S K,MOHAMMED S A,KHALAF G,et al.Role of bone marrow mesenchymal stem cells in the treatment of CCL4 Induced liver fibrosis in albino rats:a histological and immunohistochemical study[J].International Journal of Stem Cells,2014,7(2):87-97.
[10] BRUNT E M.Grading and staging the histopathological lesions of chronic hepatitis:the Knodell histology activity index and beyond[J].Hepatology,2000,31(1):241-246.
[11] 潘峥,吴性江,李为苏,等.犬肝硬化模型形成中肝功能性血流量的变化[J].中华实验外科杂志,2005,22(11):1405.
[12] BRANDAO C,FERREIRA H,PIOVESANA H,et al.Development of an experimental model of liver cirrhosis in rabbits[J].Clinical & Experimental Pharmacology & Physiology,2000,27:987-990.
[13] GEORGE J,RAO K R,STERN R,et al.Dimethylnitrosamine-induced liver injury in rats:the early deposition of collagen [J].Toxicology,2001,156(2-3):129-138.
[14] CHANG M L,YEH C T,CHANG P Y,et al.Comparison of murine cirrhosis models induced by hepatotoxin administration and common bile duct ligation[J].World Journal of Gastroenterology:WJG,2005,11(27):4167-4172.
[15] 陈刚,李宏波,邱少敏.CCl₄皮下注射制备大鼠肝纤维化模型的研究[J].现代医学,2010,38(3):225-229.
[16] BEDOSSA P,PARADIS V.Liver extracellular matrix in health and disease[J].The Journal of Pathology,2003,200(4):504-515.
[17] ATZORI L,POLI G,PERRA A.Hepatic stellate cell:a star cell in the liver[J].The International Journal of Biochemistry & Cell Biology,2009,41(8-9):1639-1642.
[18] MONTFORT I,PEREZ-TAMAYO R.Collagenase in experimental carbon tetrachloride cirrhosis of the liver[J].The American Journal of Pathology,1978,92(2):411-420.
[19] POONKHUM R,SHOWPITTAPORNCHAI U,PRADIDARCHEEP W.Collagen arrangement in space of Disse correlates with fluid flow in normal and cirrhotic rat livers[J].Microscopy Research & Technique,2014,78(2):187-193.

服务与反馈：

【文章下载】【发表评论】【查看评论】【加入收藏】

提示：您还未登录，请登录！点此登录