摘要
目的:检测肝癌干细胞标志CD133在化学诱导C57BL/6J小鼠肝癌过程中各时期的动态变化。方法:通过化学法[二乙基亚硝胺(diethylnirtosamine,DEN)/四氯化碳(carbon tetrachloride,CCl4)/乙醇]诱发50只C57BL/6J雄性小鼠肝癌,对照组为45只正常C57BL/6J雄性小鼠。观察小鼠成瘤情况和生长状态,对每2周定期处死小鼠获得的组织标本分别进行病理学切片观察,并采用实时荧光定量PCR、免疫组织化学法、蛋白质印迹法和FCM法分别检测CD133mRNA和蛋白的表达情况。结果:化学诱导20周后,成功诱发出小鼠肝癌。实时荧光定量PCR和FCM检测结果显示,第8周后诱癌组小鼠肝组织中CD133的表达高于正常对照组,差异有统计学意义(P<0.05或P<0.001);随着诱癌时间的增加,CD133的表达量呈上升趋势,第16周时诱癌组CD133的表达明显高于前期诱癌组(P<0.001)。蛋白质印迹法检测结果显示,诱癌组CD133蛋白从第4周起出现弱表达,随着诱癌时间的递增,CD133蛋白表达量逐渐增多。免疫组织化学检测结果显示,诱癌组第8周CD133出现弱阳性,第16周出现中等阳性,第20周出现强阳性;而正常对照组无表达。结论:肝癌干细胞标志CD133参与了肝癌的发生、发展全过程,随着诱癌进展其表达量逐渐增加。
Objective：To detect the dynamic change of expression of CD133, a marker of liver cancer stem cell, at different stages of liver carcinogenesis induced by chemicals in C57BL/6J mice. Methods：The tumorigenesis and the growth of chemical （diethylnirtosamine/carbon tetrachloride/ethanol）-induced primary HCC （hepatocellular carcinoma） in 50 male C57BL/6J mice were observed. Another 45 male C57BL/6J mice which had not been exposed to carcinogenic chemicals were used as the normal controls. The mice were randomly sacrificed every two weeks, and the liver tissues were removed to observe the change of pathology. The expression of CD133 mRNA was detected by RFQ-PCR （real-time fluorescent quantitative PCR）, and the expression of CD133 protein was detected by immunohistochemistry and Western blotting. The percentage of CD133-positive cells was analyzed by FCM （flow cytometry）. Results：The primary HCC was successfully induced in male C57BL/6J mice after chemical intervention for 20 weeks. The results of RFQ-PCR and FCM showed that the expression level of CD133 in the chemical-induced group was obviously higher than that in the normal control group after 8 weeks （P 〈 0.05, P 〈 0.001）. The expression level of CD133 kept on rising in the chemical-induced group as time progressed, and it was significantly higher at the 16th week than at earlier stages （P 〈 0.001）. Western blotting result showed that the CD133 weak expression could be observed at the 4th week. As time progressed, the expression level of CD133 protein was gradually increased. The result of immunohistochemistry showed that the expressions of CD133 in the chemical-induced group were weakly, moderately and strongly positive at the 8th, 16th and 20th week, respectively; while the expression of CD133 was negative in the normal control group. Conclusion：The liver cancer stem cell marker CD133 is involved in the development and progression of liver cancer, and its expression level is gradually increased in the process of carcinogenesis.
出处
《肿瘤》
CAS
CSCD
北大核心
2012年第8期585-591,共7页
Tumor
基金
广西研究生科研创新项目(编号:2011105981002M180)
