實驗性肝硬化大鼠細胞因子IL18、TNFα、IFNγ的變化及意義

1、實驗性肝硬化大鼠細胞因子IL18、TNF、IFN的變化及意義 11-01-06 11:26:00 編輯：studa20 作者：黨雙鎖，高寧，程延安，邊靜，王順達，孫明珠【摘要】 目的 探討在四氯化碳(CCl4) 復合因素誘導實驗性肝硬化大鼠的形成過程中，白介素18(IL18)、腫瘤壞死因子(TNF)、干擾素(IFN)的變化及意義。方法 將80只清潔級雄性SD大鼠隨機分為正常對照組和造模2周組、4周組、6周組，每組20只。予以CCl4復合因素誘導大鼠肝硬化，并在2、4、6周3個時間點分別處死6只大鼠。采用ELISA法檢測大鼠血清IL18、TNF、IFN和肝臟組織勻漿上清液中的IL18含量；HE染

2、色觀察肝臟的組織病理學變化。結果 CCl4復合因素誘導的實驗性大鼠，組織病理學觀察發(fā)現(xiàn)，2周時大鼠肝臟組織細胞腫脹變性，6周時有大量纖維增生，部分肝組織有假小葉的形成；隨著造模時間的延長，實驗大鼠血清IL18、TNF、IFN水平逐漸升高，造模6周組與正常對照組比較差異有統(tǒng)計學意義(P0.01)；造模組肝臟組織勻漿中IL18隨肝損害程度的加重而升高，造模6周組與正常對照組比較差異有統(tǒng)計學意義(P0.01)。結論 在CCl4復合因素誘導大鼠肝硬化的形成過程中，IL18、TNF、IFN水平逐漸升高，提示該3種細胞因子與肝硬化的形成及發(fā)展有關。 【關鍵詞】 大鼠；肝硬化；白介素18；腫瘤壞死因子；干擾

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)肝硬化是慢性肝損傷所共有的終末病理改變。在肝硬化的形成中，有大量細胞

9、因子通過參與調節(jié)組織細胞的損傷修復過程而發(fā)揮作用1。新近發(fā)現(xiàn)，白介素18(interleukin18, IL18)是新的致炎因子。研究發(fā)現(xiàn)，IL18參與了免疫調控，其過度表達可能引起免疫系統(tǒng)紊亂和組織器官的免疫炎性損害。本研究通過建立四氯化碳(CCl4)復合因素誘導的大鼠肝硬化模型，動態(tài)檢測造模組大鼠IL18及相關細胞因子腫瘤壞死因子(TNF)、干擾素(IFN)水平的變化，初步探討細胞因子在肝硬化形成中的作用及意義。1 材料與方法1.1 主要試劑及儀器CCl4(分析純)購自西安化學試劑廠；橄欖油(化學純)、膽固醇(分析純)購自上海化學試劑公司；大鼠IL18定量ELISA試劑盒為美國Biosou

10、rce公司生產,上海森雄科技有限公司進口分裝；大鼠TNF、IFN定量ELISA試劑盒購自上海西唐生物有限公司；RIPA裂解液購自北京索萊寶科技有限公司；DG3021型酶聯(lián)免疫檢測儀為華東電子管廠產品。1.2 實驗動物與分組清潔級雄性SD大鼠80只，34個月齡，體重360380g，平均(36714.12)g，購于西安交通大學醫(yī)學院實驗動物中心。據(jù)肝硬化造模時間長短將SD大鼠隨機分成4組：正常對照組、造模2周組、造模4周組、造模6周組，每組20只。所有大鼠分籠飼養(yǎng)于西安交通大學醫(yī)學院實驗動物房。正常對照組，自由取食且不做任何處理；造模2、4、6周組，每周2次皮下注射400mL/L CCl4橄欖油溶

11、液3mL/kg，首次劑量為5mL/kg；同時給予高脂飼料(80%單純玉米粉+20%豬油+0.5%膽固醇)作為唯一飲食，并以300mL/L乙醇作為飲水飼養(yǎng)動物。每周一、四注射，且注射前稱體重。于造摸2、4、6周時各組分別隨機選取動物6只，水合氯醛腹腔內麻醉，腹主動脈插管取血，并留取肝臟做組織勻漿及40g/L甲醛溶液固定，常規(guī)石蠟切片，HE染色。1.3 方法1.3.1 ELISA法測定血清IL18、TNF、IFN水平血液3000r/min離心20min，吸取血清，采用雙抗體夾心ELISA法測定，嚴格按照說明書操作。1.3.2 ELISA法測定肝組織勻漿中IL18水平取大鼠部分肝臟組織，冰冷等滲鹽水漂洗數(shù)次后，除去血液，濾紙吸干，稱取1g肝組織，用手動勻漿器將肝組