实验性肝硬化大鼠细胞因子IL18、TNFα、IFNγ的变化及意义

1、实验性肝硬化大鼠细胞因子IL18、TNF、IFN的变化及意义 11-01-06 11:26:00 编辑：studa20 作者：党双锁，高宁，程延安，边静，王顺达，孙明珠【摘要】 目的 探讨在四氯化碳(CCl4) 复合因素诱导实验性肝硬化大鼠的形成过程中，白介素18(IL18)、肿瘤坏死因子(TNF)、干扰素(IFN)的变化及意义。方法 将80只清洁级雄性SD大鼠随机分为正常对照组和造模2周组、4周组、6周组，每组20只。予以CCl4复合因素诱导大鼠肝硬化，并在2、4、6周3个时间点分别处死6只大鼠。采用ELISA法检测大鼠血清IL18、TNF、IFN和肝脏组织匀浆上清液中的IL18含量；HE染

2、色观察肝脏的组织病理学变化。结果 CCl4复合因素诱导的实验性大鼠，组织病理学观察发现，2周时大鼠肝脏组织细胞肿胀变性，6周时有大量纤维增生，部分肝组织有假小叶的形成；随着造模时间的延长，实验大鼠血清IL18、TNF、IFN水平逐渐升高，造模6周组与正常对照组比较差异有统计学意义(P0.01)；造模组肝脏组织匀浆中IL18随肝损害程度的加重而升高，造模6周组与正常对照组比较差异有统计学意义(P0.01)。结论 在CCl4复合因素诱导大鼠肝硬化的形成过程中，IL18、TNF、IFN水平逐渐升高，提示该3种细胞因子与肝硬化的形成及发展有关。 【关键词】 大鼠；肝硬化；白介素18；肿瘤坏死因子；干扰

3、素ABSTRACT: Objective To investigate the changes of interleukin18 (IL18), tumor necrosis factor(TNF) and interferon (IFN) levels of liver cirrhosis induced by the composite factors of carbon tetrachloride (CCl4) in SD rats and their significance. Methods Totally 80 male SD rats of clean class were ra

4、ndomly divided into normal control group (20 rats) and model groups, the latter of which were further divided into three groups according to the length of administration time, namely, 2week group (2 wk group), 4week group (4 wk group) and 6week group (6 wk group), with 20 rats in each. Six rats were

5、 killed after 2 wk, 4 wk and 6 wk administration time, respectively. The rat serum levels of IL18, TNF and IFN and the hepatic homogenate supernatant of IL18 were detected by ELISA; pathological changes of liver tissues were observed by HE staining. Results Pathological observation revealed that in

6、the model groups hepatic cells degenerated and swelled at week 2 while large amounts of fibrosis and pseudolobules of some liver tissues occurred at week 6. The serum levels of IL18, TNF and IFN were gradually increased with the modeling time, and they were significantly higher in 6week group than i

7、n normal control group (P0.01). The levels of hepatic homogenate supernatant of IL18 in the model groups were elevated with liver damage, and they were significantly higher in 6week group than in normal control group (P0.01). Conclusion During the formation of liver cirrhosis induced by composite fa

8、ctors of CCl4 in rats, IL18, TNF and IFN levels gradually increase, suggesting that the three cytokines play a certain role during the occurrence of liver cirrhosis in rats.KEY WORDS: rat; liver cirrhosis; interleukin18; tumor necrosis factor (TNF); interferon (IFN)肝硬化是慢性肝损伤所共有的终末病理改变。在肝硬化的形成中，有大量细胞

9、因子通过参与调节组织细胞的损伤修复过程而发挥作用1。新近发现，白介素18(interleukin18, IL18)是新的致炎因子。研究发现，IL18参与了免疫调控，其过度表达可能引起免疫系统紊乱和组织器官的免疫炎性损害。本研究通过建立四氯化碳(CCl4)复合因素诱导的大鼠肝硬化模型，动态检测造模组大鼠IL18及相关细胞因子肿瘤坏死因子(TNF)、干扰素(IFN)水平的变化，初步探讨细胞因子在肝硬化形成中的作用及意义。1 材料与方法1.1 主要试剂及仪器CCl4(分析纯)购自西安化学试剂厂；橄榄油(化学纯)、胆固醇(分析纯)购自上海化学试剂公司；大鼠IL18定量ELISA试剂盒为美国Biosou

10、rce公司生产,上海森雄科技有限公司进口分装；大鼠TNF、IFN定量ELISA试剂盒购自上海西唐生物有限公司；RIPA裂解液购自北京索莱宝科技有限公司；DG3021型酶联免疫检测仪为华东电子管厂产品。1.2 实验动物与分组清洁级雄性SD大鼠80只，34个月龄，体重360380g，平均(36714.12)g，购于西安交通大学医学院实验动物中心。据肝硬化造模时间长短将SD大鼠随机分成4组：正常对照组、造模2周组、造模4周组、造模6周组，每组20只。所有大鼠分笼饲养于西安交通大学医学院实验动物房。正常对照组，自由取食且不做任何处理；造模2、4、6周组，每周2次皮下注射400mL/L CCl4橄榄油溶

11、液3mL/kg，首次剂量为5mL/kg；同时给予高脂饲料(80%单纯玉米粉+20%猪油+0.5%胆固醇)作为唯一饮食，并以300mL/L乙醇作为饮水饲养动物。每周一、四注射，且注射前称体重。于造摸2、4、6周时各组分别随机选取动物6只，水合氯醛腹腔内麻醉，腹主动脉插管取血，并留取肝脏做组织匀浆及40g/L甲醛溶液固定，常规石蜡切片，HE染色。1.3 方法1.3.1 ELISA法测定血清IL18、TNF、IFN水平血液3000r/min离心20min，吸取血清，采用双抗体夹心ELISA法测定，严格按照说明书操作。1.3.2 ELISA法测定肝组织匀浆中IL18水平取大鼠部分肝脏组织，冰冷等渗盐水漂洗数次后，除去血液，滤纸吸干，称取1g肝组织，用手动匀浆器将肝组