药物诱导与耐药基因转染两种方法建立的人食管癌顺铂耐药细胞系的比较

目的比较药物诱导和耐药基因转染两种方法所建立的人食管癌顺铂耐药细胞系的不同特点。方法顺铂(cDDP)中等浓度、间歇作用法历时9个月建立食管癌耐药细胞系Ec9706/cDDP;同时采用脂质体转染法将人野生型DNA聚合酶β(polβ)的重组绿色荧光蛋白表达载体pEGFP-AC3转染入食管癌细胞Ec9706,经G418筛选得到稳定转染细胞系Ec9706-AC3。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。绘制生长曲线,计算群体倍增时间。RT-PCR方法检测细胞中polβmRNA的表达水平,MTT法测细胞对顺铂的敏感性,并观察冻存复苏、撤药培养对耐药性的影响。结果两种方法所获耐药细胞与亲本细胞相比形态无明显变化,polβmRNA表达均增加。Ec9706/cDDP耐药指数为15.70±1.16,冻存复苏对耐药指数影响不大,而撤药培养可使耐药性降低,细胞群体倍增时间延长;Ec9706-AC3细胞耐药指数为1.78±0.67,不受冻存复苏、撤药培养的影响,细胞群体倍增时间不变。结论用不同方法成功建立两种人食管癌顺铂耐药细胞,不同方法获得的耐药细胞生物学特性有所不同。

Comparison between human esophageal carcinoma drug-resistant cell lines established with two different methods

Objective To compare the biological characteristics of the 2 human esophageal carcinoma drug-resistant cell lines established by pulse drug exposure and transfection of DNA polymerase beta(polβ) gene.Methods The drug-resistant cell line Ec9706/cDDP was established in culture by pulse exposing Ec9706 cells to moderate concentration of cisplatin(cDDP) over a period of 9 months.The recombined enhanced green fluorescent protein(GFP) vector pEGFP-AC3 carrying wide type DNA polβ gene was transfected into Ec9706 cells by lipotransfection,and the stable transfectant Ec9706-AC3 was screened by G418.Cell growth curves were constructed for assessment of doubling time.Expression of polβ was confirmed by RT-PCR.The resistance index to cDDP was determined by the methyl thiazolyl tetrazolium assay(MTT).Results There was no significant morphologic difference between the 2 established human esophageal carcinoma drug-resistant cell lines,in which polβ expression level was significantly increased.The resistant index of Ec9706/cDDP was 15.70±1.16 and maintained after frozen resuscitation,but reduced after cultured without cDDP for 1 month.The resistant index of Ec9706-AC3 was 1.78±0.67 and not affected by frozen resuscitation or cDDP withdraw.Althoug doubling time of Ec9706/cDDP was prolonged,that of Ec9706-AC3 was not affected.Conclusion Two human esophageal carcinoma drug-resistant cell lines were established by two different methods,presenting different biological characteristics.