黄曲霉毒素诱发大鼠肝细胞癌过程中细胞外调节蛋白激酶信号转导通路的动态变化

Dynamic changes of signal transduction pathway of extracellular signal regulated kinase during hepatocarcinoge-nesis induced by aflatxin B1 in rats

目的:探讨细胞外信号调节蛋白激酶(extracellular signal regulated kinase,ERK)信号转导通路在肝细胞癌(hepato-cellular carcinoma,HCC)发生、发展过程中的变化及其作用。方法:以SD大鼠为研究对象,随机分为空白对照组和实验组,实验组腹腔注射黄曲霉毒素B1(aflatoxin B1,AFB1),分别于实验第12、20、36和46周进行肝活检,58周时处死大鼠并剖腹获取肝组织及肿瘤组织,对所有标本均进行常规病理组织学检测后,采用RT-PCR和Western印迹法分别检测所获取组织中ERK1和ERK2 mRNA及蛋白的表达。结果:实验组46周时发现首只罹患HCC的大鼠,实验结束时实验组共有30只大鼠存活,其中24只大鼠发生HCC,6只大鼠无任何肿瘤发生;空白对照组11只大鼠均无肿瘤发生。RT-PCR检测发现,发生肿瘤、未发生肿瘤及空白对照组大鼠肝组织在实验不同时期的ERK1和ERK2 mRNA的表达水平无明显差异(P>0.05);但发生肿瘤大鼠的HCC组织ERK1和ERK2 mRNA表达水平较发生肿瘤前各时间点活检肝组织有明显上升(P<0.01),且较癌旁肝组织、同期无癌大鼠及空白对照组大鼠肝组织的表达明显上调(P<0.05)。Western印迹法检测结果发现,HCC组织p-ERK1及p-ERK2蛋白的表达较癌旁肝组织、发生肿瘤前各时期肝组织及无癌大鼠肝组织明显上升(P<0.01)。结论:在HCC发生早期ERK1和ERK2处于相对正常的状态,而在癌变期ERK1和ERK2信号转导通路表现出异常激活状态,提示ERK1和ERK2信号转导通路可能参与了HCC的发生与发展。

Objective：To explore the changes of signal transduction pathway of extracellular signal regulated kinase（ERK1/2） in the initiation and progression of hepatocellular carcinoma（HCC） and their significance.Methods：Totally 77 male SD rats were randomly divided into two groups.The experimental group（n=66 rats） were administered aflatoxin B1（AFB1） by intraperitoneal injection for 32 weeks.Whereas,11 rats were not administered AFB1 and served as control group.All of animals underwent liver biopsy at week 12,week 20,week 36,and week 46 during the experiment.All the surviving rats were sacrificed at week 58 and their livers and/or tumor tissues were isolated.Routine Mayer＇s hematoxylin and eosin（H＆E） staining was performed on all the livers and tumor samples.The mRNA and protein expression levels of ERK1/2 genes were determined by RT-PCR and Western blotting,respectively.Results：The first case of HCC was discovered at week 46.Totally 30 rats survived at the end of the experiment（week 58）.Among the AFB1-treated rats 24 rats developed HCC and 6 rats had no carcinoma.No tumor was developed in the control group.RT-PCR showed that there was no significant difference in ERK1 and ERK2 mRNA expression in liver tissues at different stages between hepatocarcinogenesis group,non-hepatocarcinogenesis group,and control group（P〉0.05）,but the expression levels of ERK1 and ERK2 mRNA in HCC tissues were higher than those in biopsy tissues before HCC development（P〈0.01） and up-regulated compared with para-HCC liver tissues,liver tissues in rats without hepatogenesis,and normal liver tissues in control rats（P〈0.05）.Western blotting showed that the expression levels of p-ERK1 and p-ERK2 proteins in HCC tissues were higher than those in para-HCC tissues,liver tissues in rats without hepatogenesis,and biopsy tissues before HCC development（P〈0.01）.Conclusion：The data show that the ERK1 and ERK2 signal transduction pathway is in relatively normal condition before HCC development,and ERK1 and ERK2 signaling transduction pathway is abnormally activated in the progression stage of HCC.The results indicate that the ERK1/2 signaling transduction pathway is involved in the initiation and progression of HCC.