小剂量二乙基亚硝胺诱导大鼠肝纤维化模型的建立

doi:10.3969/j.issn.1672-5069.2019.01.010

摘要/Abstract

摘要： 目的探讨采用小剂量二乙基亚硝胺（DEN）诱发大鼠肝纤维化实验性模型的方法。方法取Wistar大鼠30只,随机分为模型组和正常组,每组15只。给予模型组DEN溶液10 mg·kg^-1灌胃,1次/d。在10周后,处死动物,取肝组织行病理学检查和细胞超微结构观察。测定血清超氧化物歧化酶（SOD）、丙二醛（MDA）、谷胱甘肽过氧化物酶（GSH-Px）含量。结果模型组动物血清ALT和AST水平分别为（410.1±93.6） U/L和（399.7±90.9） U/L,显著高于对照组【（56.51±11.2） U/L 和（63.26±10.0）U/L,P<0.001】,模型动物血清SOD、GSH-Px和MDA活性分别为（83.08±22.0) U/mg、（24.65±2.0） U/mg和（9.78±2.0) nmol/mg,与对照组比,均有显著性差异【（178.57±22.8）U/mg、（45.16±2.9）U/mg和（3.76±0.7）U/mg,P<0.01】;模型组动物肝组织肝细胞变性、增生、坏死和假小叶形成,超微结构改变以肝细胞核内异染色质增加、细胞器数量减少、基质密度降低和肝糖元减少表现为主。结论采用小剂量DEN能成功诱导大鼠肝纤维化模型,造模成功率高,病变稳定。

关键词: 肝纤维化, 二乙基亚硝胺, 超微结构, 大鼠

Abstract: Objective To establish liver fibrosis model with low-dose diethylnitrosamine（DEN） in rats. Methods Thirty Wistar rats were randomly divided into model group and control group with 15 in each. The rats in the model group were treated with low-dose DEN（10 mg·kg^-1 body weight,orally,once daily） for 10 weeks. After sacrificing,the hematoxylin and eosin staining was performed to determine histopathological alterations and transmission electron microscopy（TEM） was used to observe the changes of ultrastructures in the hepatic cells. The automatic biochemical analyzer was used to measure ALT and AST levels,and biochemical methods was used to determine the content of serum SOD,MDA and GSH-Px. Results The levels of ALT and AST in the model group were（410.08±93.6） U/L and（399.7±90.9） U/L,respectively,significantly higher than those in the control group [（56.51±11.2） U/L and（63.26±10.0） U/L,P<0.001];serum contents of SOD,GSH-Px and MDA in the model group were（83.08±22.0） U/mg,（24.65±2.0） U/mg and （9.78±2.0） nmol/mg,respectively,significantly lower than those in the control group[（178.57±22.8） U/mg,（45.16±2.9） U/mg and（3.76±0.7） U/mg,P<0.01];the histological alterations of liver cells in the model group were mainly hepatic deforming,proliferation,necrosis and pseudolobule formation and the ultrastructural changes were mainly the increase of heterochromatin in the nucleus,decrease of organelle,matrix density and hepatin. Conclusion Low-dose DEN could successfully induce liver fibrosis and cirrhosis in rats with a high successful rates and stable pathological changes.

Key words: Liver fibrosis, Diethylnitrosamine, Ultrastructure, Rats

玉苏甫·吐尔逊, 赵燕霞. 小剂量二乙基亚硝胺诱导大鼠肝纤维化模型的建立[J]. 实用肝脏病杂志, 2019, 22(1): 33-36.

Yusup Tursun, Zhao Yanxia.. Establishment of low-dose diethylnitrosamine-induced liver fibrosis model in rats[J]. JOURNAL OF PRACTICAL HEPATOLOGY, 2019, 22(1): 33-36.

参考文献

[1] Clevenger CV.Roles and regulation of stat family transcription factors in human breast cancer. Pathology,2004,165(5):1449-1460.
[2] 仝艳艳,李光明,邓怡林,等. 氧化苯砷体外对大鼠原代肝星状细胞活化的阻抑作用研究. 实用肝脏病杂志,2016,19(4):413-415.
[3] Rocchi P,Beraldi E,Ettinger S,et al.Increased Hsp27 after androgen ablation facilitates androgen-independent progression in prostate cancer via signal transducers and activitors of transcription3-mediated suppression of apoptosis,Cancer Res,2005,65(23):11083-11093.
[4] Zhang X,Zhang J,Wei H,et al.STAT3-decoy oligodeoxynucleotide inhibits the growth of human lung cancer via down-regulating its target genes. Oncol Rep,2007,17(6):1377-1382.
[5] Sells,Dunsford H A. Evidence for the stem cell origin of hepatocellular carcinoma and cholangiocarcinoma. Am J Pathol, 1989,134(6):1347-1363.
[6] Isbrucker RA,Peterson TC.Platelet-derived growth factor and pentoxifylline Modulation of synthesis in myofibroblasts. Toxicol Appl Pharmacol.1998,149(1):120-126.
[7] 付指辉,李三强,丁康熙,等.四氯化碳诱导的肝纤维化小鼠肝组织VEGF表达变化. 实用肝脏病杂志,2016,19(1):81-82.
[8] Ito H,Hamerman JA.TREM-2,triggering receptor expressed onmyeloid cell-2,negatively regulates TLR response indendritecells.Eur J Immunol,2012,42:176-185.
[9] 戴光荣,谭玉娥,刘文娜,等.Logistic回归分析食管静脉曲张破裂出血的危险因素. 实用肝脏病杂志,2015,18(3):387-389.
[10] 王军,彭浩.特利加压素联合生长抑素治疗肝硬化食管胃底静脉曲张破裂出血疗效观察.实用肝脏病杂志,2015,18(4):418-419.
[11] 刘文娜,宁涛,戴光荣,等.多层螺旋CT门静脉造影与B超检查预测肝硬化食管静脉曲张出血的对比研究.实用肝脏病杂志,2015,18(1):47-49.
[12] 张海燕,温韬,卢静,等.四氯化碳诱导大鼠慢性肝损伤模型方法的探讨.实用肝脏病杂志,2009,12(3):161-224.
[13] 刘莺,刘平,王磊.扶正化癖方对肝纤维化大鼠不同病理阶段肝组织蛋白质组变化的影响. 中华肝脏病杂志,2006,14(6):422-425.
[14] Piao RL,Brigstock DR,Zhu J,et al.Clinical significance of connective tissue growth factor in hepatitis B virus-induced hepatic fibrosis. World J Gastroenterol,2012,18(18):2280-2286.
[15] 胡景峰,李三强,吴秦川.二乙基亚硝胺诱导肝癌小鼠肝组织胶原纤维的变化.实用肝脏病杂志,2015,18(5):521-523.
[16] Hernandez-Gea V,Friedman SL.Pathogenesis of liver fibrosis. Annu Rev Pathol Mech,2011,6:425-456.
[17] Yovchcv MI,Xue Y,Shafritz DA,et al.Repopulation of the fibrotic/cirrhotic rat liver by transplanted hepatic stem/progenitor cells and mature hepatocytes. Hepatology,2014,59:284-295.
[18] Wang P,Liu T,Cong M,et al.Expression of extracellular matrix genes in cultured hepatic oval cells: an origin of hepatic stellate cells through transforming growth factor beta.Liver Int,2009,29:575-584.
[19] Yang AT,Hu DD,Wang P,et al.TGF-β1 induces the dualregulation of hepatic progenitor cells with both anti-and proliver fibrosis. Stem Cells Int,2016,2016:149-694.
[20] Sato R,Maesawa C, Fujisawa K,et al.Prevention of critical telomere shortening by oestradiol in human normal hepatic cultured cells and carbon tetrachloride induced rat liver fibrosis. Gut,2004,53(7):1001-100.
[21] Roth-Eichhorn S,Kuhl K,Gressner AM.Subcellular localization of(latent) transforming growth factor beta and the latent TGF-beta binding protein in rat hepatocytes and hepatic stel-late cells. Hepatology,1998,28(6):1588-1596.
[22] Fukumasu H,Avanzo JL,Heider R,et a1. Prolective effects of guarana(Panllinia cupana Mart.var.Sorbilis) against DEN-induced DNA damage on mouse liver. Food Chem Toxicol,2006,44(6):862-867.
[23] Chen Q,Gong B,Almasan A.Distinct stages of cytochrome C release from mitochondria:evidence for a feedback amplification loop linking caspase activation to mitochondrial dysfunction in genotoxic stress induced apoptosis. Cell Death Differ,2000,7(2):227-233.
[24] Vernon G,Baranova A,Younossi ZM.Systematic review: the epidemiology and natural history of non-alcoholic fatty liver disease and non-alcoholic steatohepatitis in adults. Aliment Pharmacol Ther,2011,34:274-285.