Fas／Fas配体系统及其下游信号转导通路的激活促进酒精性肝炎及酒精性肝纤维化的进展  

Activation of Fas/FasL and its downstream signaling pathway promotes development of alcoholicsteatohepatitis and liver fibrosis in mice

作　　者：任伟光[1] 孔令波[1] 米红梅[1] 赵素贤[1] 张玉果[1] 王荣琦[1] 南月敏[1] 

机构地区：[1]河北医科大学第三医院中西医结合肝病科,石家庄050051

出　　处：《中华肝脏病杂志》2013年第2期129-133,共5页Chinese Journal of Hepatology

基　　金：中国肝炎防治基金会王宝恩肝纤维化基金（2009009）

摘　　要：目的探明Fas／Fas配体（FasL）系统及其主导信号转导通路在酒精性肝炎和肝纤维化发生、发展中的作用及其机制。方法C57BL／6J小鼠以含4％（V／叨乙醇的Lieber-DeCarli液体饲料喂养4周，自第5周起联合微量cck腹腔注射至第8周，分别建立酒精性肝炎和肝纤维化模型。脱氧核糖核昔酸末端转移酶介导的缺口末端标记法（TUNEL）检测肝细胞凋亡。分别采用逆转录一聚合酶链反应、Westernblot及免疫组织化学染色检测肝组织Fas、FasL、天冬氨酸特异性半胱氨酸蛋白酶3（caspase3）及细胞色素P4502El（CYP2E1）的mRNA及蛋白质表达。多组样本均数间差异比较用单因素方差分析或Kmskal-WallisH检验，组间比较用LSD—f检验或Mann-WhitneyU检验。结果应用含4％（V／叻乙醇的Lieber-DeCarli液体饲料联合微量cch腹腔注射可快速建立小鼠酒精性肝炎和肝纤维化模型。肝细胞凋亡数随肝脏炎症及纤维化的加重而增高，并伴有凋亡相关基因表达增强。对照组、酒精性肝炎组、酒精性肝纤维化组小鼠细胞凋亡相关基因表达水平依次升高：FasmRNA的相对表达量分别为0．50±0．05、0．61±0．10、0．76±0．03（H=12．137P〈0．05），Fas蛋白的相对表达量分别为0．52±0．14、0．86±0．10、0．99±0．09（F=12．758P〈0．01）；FasLmRNA相对表达量分别为0．31±0．03、0．53±0．02、1．02土0．04（F=153．260P〈0．01）；caspase3mRNA对表达量分别为0．86±0．11、0．85±0．05、1．33士0．16∽=8．740，P〈0．01），caspase3蛋白相对表达量分别为0．40±0．03、0．69±0．06、1．02±0．10（F=90．785，P〈0．01）；CYP2ElmRNA相对表达量分别为0．72±0．14、1．00±0．15、1．30±0．20（F=4．713，P〈0．01）；各组问比较，尸值均〈0．05。免疫组织化学染色结果显示，FasL及CYP2El的蛋白与mRNA的表达趋势一致。结论Fas／FasL系统及其下游信号转导通路的�Objective To explore the role and mechanism of the Fas/Fas ligand （FasL） system and its downstream signaling pathway related to the progression of alcoholic steatohepatitis and liver fibrosis. Methods Eighteen C57BL/6J mice were randomly divided into three groups： controls; alcoholic steatohepatitis model, given four-weeks of a 4% ethanol-containing Lieber-DeCarli liquid diet; alcoholic steatohepatitis and liver fibrosis model, given the four-week alcohol diet followed by twice weeklyintraperitoneal injections of carbon tetrachloride （5% olive oil solution; 2 mL/kg dose） during the fifth to eighth weeks. Mice in the model groups were sacrificed at the end of week 4 and 8, respectively, along with control mice for comparative analyses. Liver tissue sections were evaluated for hepatocellular apoptosis by terminal deoxynucleotidyl transferase dUTP nick-end labeling （TUNEL） assay. The mRNA expression of Fas, FasL, cysteine aspartate-specific proteases 3 （caspase 3）, and cytochrome P450 2El （CYP 2El） in liver tissues was detected by reverse transcription （RT）-PCR, visualized by ethidium bromide staining, and normalized to the gray-value of GAPDH expression. The protein expression of Fas and caspase 3 were detected by western blotting （b-actin normalized）, and of FasL and CYP 2El by immunohistochemistry staining. Intergroup differences and statistical significance were evaluated by single factor analysis of variance and the least squares difference-t test or the Kruskal-Wallis H test and the Mann-Whitney U test. Results The number of apoptotic cells in the liver sections was significantly higher in both model groups with alcoholic steatohepatitis （vs. controls） and the amount in the alcoholic steatohepatitis plus liver fibrosis model was significantly higher than that in the model with only alcoholic steatohepatitis. In addition, activation of Fas, FasL and its downstream signaling pathway showed an increasing trend with extent of liver injury. The hepatic mRNA （by RT-PCR） and prot

关 键 词：肝炎 酒精性 肝硬化 细胞凋亡 炎症 Fas FAS配体 

分 类 号：R575.2[医药卫生—消化系统]