稳定高表达人野生型DNApolβ食管癌细胞Ec9706的生物学特征

Biological characteristics of Ec9706 cells stably overexpressing human wild type DNA polβ gene

目的:建立稳定高表达人野生型DNA聚合酶beta(polβ)的食管癌细胞系Ec9706,并对该细胞系的生物学特征进行分析。方法: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,荧光显微镜观察转染效果,经G418筛选得到稳定转染细胞系。RT-PCR方法检测转染细胞polβmRNA的表达水平,绘制生长曲线,软琼脂实验测细胞克隆形成率,MTT法测转染前后细胞对顺铂的敏感性。结果:荧光显微镜下转染细胞荧光强,转染效率高,RT-PCR结果表明转染细胞的polβ表达较空载体转染细胞、对照细胞增加。转染前后细胞生长速度接近,转染细胞周期分布发生改变,多被阻滞在S期,在软琼脂实验中克隆形成率增加,对顺铂的敏感性降低。结论:成功建立了稳定高表达人野生型polβ的Ec9706细胞系,polβ的高表达与细胞的细胞周期分布、恶性增殖能力及耐药性有一定关系。

Objective：To establish human esophageal carcinoma cell line Ec9706 stably overexpressing human wide type DNA polymerase beta gene （polβ） ,and investigate its biological characteristics. Methods： A recombined enhanced green fluorescent protein （GFP） vector pEGFP - AC3 carrying wide type DNA poll3 gene was transfected into Ec9706 cells by lipotransfection, and the stable transfectants were screened by G418. Stable overexpression of polβ in Ec9706 was confirmed by RT - PCR. Cell growth curve was painted. Flow cytometry （FCM） was performed to determine cell cycle. Clones forming rate was determined by cell soft agar test. The resistance index to cDDP was determined by the methyl thiazolyl tetrazolium assay （MTT）. Results： The vector pEGFP - AC3 was transfected into Ec9706 cells successfully. Expression level of polβ in the transfected cell increased significantly. The proliferation speed did not change. The cell cycle distribution changed and most cells remained at S phage. And there was significant change in soft agar growths. Clones forming rate of transfected cell increased. And sensitivity to cDDP decreased. Conclusion：Ec9706 cell line stably overexpressing human wide type DNA polβ has been established successfully. Overexpression of polβ has some effect on the cell cycle distribution, malignant proliferation ability and drug resistance.