胆道感染与肝细胞凋亡及凋亡相关基因的表达

Cholangitis and hepatocyte apoptosis and expression of apoptosis gene

目的探讨胆道感染时是否发生肝细胞凋亡,胆道感染时间与肝细胞凋亡的关系及胆道感染时凋亡相关基因的表达,为胆道感染性疾病提供基础病理认识。方法选用日本大耳白兔65只,随机分成对照组5只,实验组60只。实验组胆总管注入1mL浓度为1×106的埃希氏大肠杆菌,制成急性胆道感染动物模型。实验组动物于注入大肠杆菌后6、12、24、48、72和96h处死。对照组动物胆总管内注入1mL生理盐水。取各组动物组织,甲醛液固定后制成蜡块。每组肝组织切片作HE染色,光镜下观察肝组织的病理改变。每组肝组织切片行TUNEL试验,检测调亡肝细胞;免疫组织化学试验检测凋亡相关基因bcl-2,Bax的表达。结果对照组可观察到极少量凋亡细胞。实验组早期(6￣12h)即可发现较多凋亡肝细胞,中期(24￣48h)可发现大量凋亡肝细胞,晚期(72￣96h)凋亡肝细胞持续增多。凋亡指数在各时相段之间差异有显著性(P<0.01),实验组和对照组比较差异也有显著性(P<0.01)。bcl-2,Bax表达检测结果:bcl-2在对照组和实验组早期有少量表达,在实验组中、晚期表达不明显,bcl-2的阳性率在各组之间差异无显著性(P>0.05)。Bax的阳性率在对照组和实验组之间差异有显著性(P<0.05)。实验组:6h组与12h组比较,差异无显著性(P>0.05),6h组分别与48、72、96h组比较,差异有显著性(P<0.05),12h组与24h比较,差异无显著性(P>0.05),12h组分别与48、72、96h比较,差异有显著性(P<0.05),24h组与48、72、96h组间分别比较,差异无显著性(P>0.05)。结论胆道感染时,不同时相段肝细胞均出现凋亡。细胞凋亡的出现早于细胞坏死,且随着胆道感染时间延长,肝细胞凋亡指数不断增加。表明细胞死亡早期以凋亡为主,凋亡的发生贯穿整个病理过程。胆道感染时,诱导凋亡基因Bax表达增强,抑制凋亡基因Bcl-2表达减弱,表明基因调控机制在胆道感染时诱发肝细胞凋亡中发挥重要作用。

【Objective】 To study whether the hepatocyte apoptosis can be induced by cholangitis. To explore the relationship between the hepatocyte apoptosis and the time of cholangitis. To investigate the expression of apoptosis gene. 【Methods】 65 rabbits were divided randomly into control group （n=5） and experimental group （n=60）. Cholangitis models were by injected 1ml Escheriehia coli （1×10^6 CFU/mL） into the rabbits＇ choledochoducts. 10 rabbits were killed respectively at the time point of 6, 12, 24, 48, 72, 96 h. In control group rabbits, 1 mL normal saline was injected into the ehotedochoducts insteadly; control group animals were killed at time point of 24 h. The liver tissues were removed, fixed by Formaldehyde. Stained the tissue sections by HE method, obse,wed the histologic changes, Apoptosis was detected by TUNEL method, the gene of bcl-2, Bax related to apoptosis were detected by immunohistochemical technique. 【Results】 TUNEL test： Fewer apoptosis cells and nerosis cells were observed in the control group hepatocyte; more apoptosis cells, few nerosis cells were observed in the experimental group hepatocyte at early period （6h-12h）; lots of apoptosis cells and more nerosis cells were observed at ntiddle period （24～48 h）; apoptosis cells and nerosis cells increased to tile later periol （72～96 h）. Apoptosis index was different significantly between different experiential group （P 〈 0.01）, contrul group and experiential group （P 〈 0.01）. bcl-2, Bax test： the positive rate of the bcl-2 expressions had no significant difference arnong groups （P 〉 0.05）; there were obvious difference between control group and experimental group （P 〈 0.05）. Compared with the Box expression at 6 or 12 h, significant difference were found for the time of 48, 72, 96 h （P 〈 0.05）, respectively. 【Conclusions】 Hepocyte apoptosis occurred when there was cholangitis. Apoptosis occurred earlier than necrosis and inereased with the infection progressing. The expression of Box was strengthened when cholangitis happened, which suggested that the gette related to apoptosis played a great role in inducing apoptosis.