刀豆蛋白A诱导小鼠肝纤维化动物模型的建立

The immune hepatic fibrosis model induced by repeatedly injection of concanavalin A in Kunming mice

目的利用刀豆蛋白A建立昆明小鼠肝纤维化模型并探讨其机制。方法昆明小鼠210只,随机分为7组,正常A组(注射等量生理盐水),刀豆蛋白A10mg每周1～3次(分别为B、C、D组)、15mg每周1～2次(分别为E、F组)、20mg每周1次(G组),采用尾静脉注射法攻击昆明小鼠,分别在第1周、第5周、第12周和第20周处死3只小鼠,留取肝组织标本行苏木精-伊红染色、纤维Masson染色,并行Knodell HAI和Ishak肝纤维化半定量计分,最后一次留取血清标本,检测血清IL-18、TNF-α和IFN-γ。结果 15mg每周1次,10mg每周2次组实验早期肝脏组织以坏死为主;实验中期(5～12周)坏死减轻,细小纤维条索出现;实验晚期(20周)纤维间隔逐渐增宽。10mg每周1次组实验晚期(20周)纤维条索形成,纤维间隔有增宽趋势。10mg每周3次组和20mg每周1次组全部死亡前,未发现有明显的肝纤维化形成。肝组织炎症活动度半定量计分显示刀豆蛋白A注射次数和注射剂量较大组评分较高(P<0.05)。肝纤维化半定量计分显示10mg每周2次组较15mg每周1次,10mg每周1次组高(P<0.05)。各组注射刀豆蛋白A后均出现了IL-18、TNF-α和IFN-γ的升高,并且IL-18和TNF-α表现出相关性(r=0.889,P<0.05)。结论利用刀豆蛋白A建立昆明小鼠的肝纤维化模型,其形成机制与IL-18、TNF-α和IFN-γ的升高密切相关。

Objective To creat the immune hepatic fibrosis model induced by repeatedly injection of concanavalin A in Kunming mice and explore the mechanisms.Methods Kunming mice were divided into seven groups,control group mice(Ga) were injected weekly with normal saline,concanavalin A groups were divided into Gb,Gc,Gd,Ge,Gf and Gg.Gb mice were injected with concanavalin A(10 mg/kg) once a week;Gc mice were 10 mg/kg twice a week;Gd mice were 10 mg/kg three times a week;Ge mice were 15 mg/kg once a week;Gf mice were 15 mg/kg twice a week;Gg mice were 20 mg/kg once a week.Twenty-four hours after concanavalin A challenge at 1,5,12 and 20 weeks.Three mice were killed by cervical dislocation,respectively.The blood sera were storaged at 4 ℃ for checking up IL-18,TNF-α and IFN-γ.The liver of different groups mice were excised and fixed in 10 % formalin for HE staining and Masson staining or frozen in liquid nitrogen for immunohistochemical staining for IL-18.Results At the beginning of experiment(1-5 weeks),hepatocellular necrosis had become widespread throughout the lobule.No evidence of hepatic fibrosis was observed at this period.However,at middle of experimemt(6-12 weeks),the small fibrosis strip had been found in Gb,Gc,Gd,Ge,bridging fibrosis and large fibrosis strip in the parenchyma with hepatocellular necrosis was detectable in Gb,Gc,Ge at 20 weeks by HE staining ?and were higher than control group(P <0.05).Moreover,there were relation between IL-18 and TNF-α(r = 0.889,P < 0.05).Conclusions The immune hepatic fibrosis model could be induced by repeatedly injection of concanavalin A in Kunming mice and the mechanisms releated with IL-18,TNF-α and IFN-γ.