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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.
OBJECTIVE: To establish a cisplatin-resistant human esophageal cancer cell line and to overexpress human wild-type DNA polymerase beta (polβ) esophageal cancer cell lines and analyze the correlation between polβ overexpression and esophageal cancer cell resistance.Methods: (cDDP) at medium concentration for 9 months. The resistant cell line Ec9706 / cDDP was established by intermittent treatment for 9 months. At the same time, the recombinant plasmid pEGFP-AC3 expressing human wild-type polβ was transfected into Ec9706 cells by lipofectamine transfection. The transfected cell lines were stained by fluorescence microscopy.The morphological changes of cells were observed by inverted microscope.RT-PCR was used to detect the expression of polβmRNA in drug-resistant cells and transfected cells respectively.MTT assay was used to detect the expression of polD mRNA Sensitivity.Results: The fluorescence of transfected cells under fluorescence microscope was strong, the transfection efficiency was high, the morphological changes of drug-resistant cells before and after transfection showed no significant changes under inverted microscope.RT-PCR results showed that the expression of polβ in Ec9706 / cDDP was higher than that in transfected cells The expression of polβ in transfected cells was higher than that in transfected cells and the number of control cells was also increased.The drug resistance index of Ec9706 / cDDP cells was 15.70.The sensitivity of cells to cDDP was decreased after transfection, The number was 1.78.Conclusion: The Ec9706 / cDDP cell line was successfully established and Ec9706 cell line stably overexpressing human wild-type polβ was overexpressed and the expression of polβ in drug-resistant cells was also increased There is some correlation between the expression of .polβ and the drug resistance of esophageal cancer cells.