摘要
目的:建立人食管癌顺铂耐药细胞系及稳定高表达人野生型DNA聚合酶beta(polβ)的食管癌细胞系,分析polβ高表达与食管癌细胞耐药的相关性.方法:顺铂(cDDP)中等浓度、间歇作用法历时9mo建立耐药细胞系Ec9706/cDDP;同时脂质体转染法将人野生型polβ重组绿色荧光蛋白表达载体pEGFP-AC3转染入Ec9706细胞,经G418筛选得到稳定转染细胞系.荧光显微镜观察转染效果,普通倒置显微镜观察细胞形态变化.RT-PCR方法分别检测耐药细胞与转染细胞中polβmRNA的表达水平,MTT法检测各组细胞对cDDP的敏感性.结果:荧光显微镜下转染细胞荧光强,转染效率高,倒置显微镜下耐药细胞与转染前后细胞形态无明显改变.RT-PCR结果表明耐药细胞Ec9706/cDDP中polβ表达较亲本细胞增加,转染细胞的polβ表达较空载体转染细胞、对照细胞也增加.Ec9706/cDDP细胞耐药指数为15.70;转染后细胞对cDDP的敏感性降低,耐药指数为1.78.结论:成功建立了耐药细胞系Ec9706/cDDP与稳定高表达人野生型polβ的Ec9706细胞系,polβ的高表达可引起耐药性的产生,耐药细胞中polβ的表达也增高.polβ的表达与食管癌细胞的耐药性有一定相关性.
AIM： To establish a cisplatin-induced human esophageal carcinoma drug-resistant cell line Ec9706/cDDP and human esophageal carcinoma cell line Ec9706 stably overexpress- ing human wide-type DNA polymerase beta gene（polβ） ,and then investigate the correlation between overexpression of polβ gene and drug resistence of human esophageal carcinoma cells. METHODS： The drug-resistant cell line Ec9706/cDDP was established in culture by pulse exposure of Ec9706 cells to moderate concentration of cisplatin （cDDP） over a period of 9 months. A recombinant enhanced green fluorescent protein （GFP） vector pEGFP-AC3 carrying wide-type DNA polβ gene was transfected into Ec9706 cells by lipotransfection, and the stable transfectants were screened by G418. Expression of polβwas confirmed by RTPCR. The resistance index to cDDP was determined by the MTY assay. RESULTS ： Under fluorescence microscope, the transfected ceils showed strong green fluorescent light. Under inverted microscope, cell morphology in every group didn＇t change. Expression level of polβ in the Ec9706/cDDP and the transfected cells increased significantly than that in the controls. The resistance index of the Ec9706/cDDP and the transfected ceils was 15.70 and 1.78, respectively. CONCLUSION： A cisplatin-induced human esophageal carcinoma drug-resistant cell line Ec9706/cDDP and Ec9706 ceil line stably overexpressing polβ gene have been established successfully. Expression level of polβincreases in drug-resistant cells, and overexpression of polβ can result in drug resistence. There is an association between overexpression of polβ and drug resistence of human esophageal carcinoma cells.
出处
《第四军医大学学报》
CAS
北大核心
2007年第12期1065-1068,共4页
Journal of the Fourth Military Medical University
基金
国家自然科学基金(39870287)
关键词
DNA聚合酶Β
转染
食管肿瘤
肿瘤细胞
培养的
抗药性
肿瘤
四甲基偶氮唑蓝
DNA polymerase beta（polβ）
transfection
esoph-ageal neoplasms
tumor cells, cultured
drug-resistance, neoplasm
methyl thiazolyl tetrazoliumassay（MTI＇）
