食管癌耐阿霉素EC9706中干细胞富集及上皮间质化

摘要

目的:建立耐阿霉素(ADM)食管癌细胞株,命名为EC9706/ADM,探讨该细胞株的上皮间质化(EMT)及肿瘤干细胞富集现象.方法:用中等浓度间歇法建立食管癌耐阿霉素细胞株,MTT法测定细胞株对ADM、5-FU、顺铂、环磷酰胺的耐药指数.流式细胞仪检测EC9706/ADM及EC9706中CD44+CD24-细胞比例,MTT法、Transwell、平板克隆形成和球囊培养实验分别检测耐药对细胞增殖、迁移与转移、体外成瘤和克隆形成能力的影响;荧光定量PCR检测EC9706/ADM干细胞标志ALDH1、CD133及Oct4及EMT相关分子标志物E-钙黏蛋白及间质细胞分子标记N-钙黏蛋白、波形蛋白、纤维连接蛋白的表达.结果:EC9706/ADM的耐药指数为17.8,对5-FU、顺铂、环磷酰胺产生了交叉耐药性.与EC9706细胞相比,EC9706/ADM耐药细胞转移能力明显增强,表现出更高的微球体形成能力,E-cadherin表达降低而Fibronectin表达显著增高.结论:食管癌耐阿霉素细胞EC9706/ADM发生了EMT现象,转移迁移能力增强,细胞表现出更高的干细胞特性,化疗药物有助于肿瘤干细胞的富集.%Objective:To investigate the characteristic of the cell invasion ability of breast cancer cells with ac-quired resistance to chemotherapy which named EC9706/ ADM.Comparing the proportion of cancer stem cells between the EC9706/ ADM and its original parent cell line EC9706 in vitro.Methods:To establish a human adriamycin-re-sistant ESCC cell line by middle doses of intermittent induction,the drug resistance of EC9706/ ADM was estimated by MTT method.To detected the rate of CD44 + CD24-cells in the EC9706 and EC9706/ ADM cells.The biological behavior of EC9706/ ADM subclones examined included cell proliferation,colony-forming assay,sphere culture and metastasis in vitro.The mRNA that the EC9706 and EC9706/ ADM cells expression levels of the selected genes (E-cadherin,N-cadherin,Vimentin,Fibronectin,ALDH1,CD133 and Oct4)were validated with real-time quantitative RT-PCR.Results:The drug resistant index of EC9706/ ADM cell kept on 17.8 times over EC9706 cell.EC9706/ADM had obvious cross-resistance to other chemotherapy drugs (cis-platinum,5-FU,cyclophosphamide).The a-bility of cell colony-forming assay,sphere culture and invasion was enhanced significantly in EC9706/ ADM com-pared to EC9706 cells.EC9706/ ADM cells expressed increased levels of mRNA for Fibronectin,ALDH1 and Oct4 but decreased the level of E-cadherin.Conclusion:EC9706/ ADM cells exhibited epithelial-mesenchymal transition phenotype and stem cell like characteristics.EC9706/ ADM has a higher proportion of cancer stem cells than its origi-nal parent cell line EC9706 in vitro.