人食管癌顺铂耐药细胞系Ec9706/cDDP的建立及其生物学特征

Establishment of a cisplatin-induced human esophageal carcinoma drug-resistant cell line and its biological characteristics

目的:建立人食管癌顺铂耐药细胞系Ec9706/cDDP,探讨其生物学特征.方法:采用顺铂(cDDP)中等浓度、间歇作用方法历时9mo建立耐药细胞系Ec9706/cDDP,采用MTT法测定其对cDDP的耐药指数,观察冻存、撤药对耐药性的影响,比较耐药与亲本细胞生长曲线、群体倍增时间及贴壁率的不同,流式细胞仪测细胞周期,软琼脂实验测细胞的恶性增殖能力,罗丹明实验测其对药物的摄入、排出能力.结果:人食管癌顺铂耐药细胞系Ec9706/cDDP的耐药指数为15.7,冻存对耐药性影响不大,撤药培养可使耐药性降低,耐药细胞群体倍增时间延长(29.79±0.48hvs25.79±0.45h,P<0.05),生长缓慢,G0/G1、S期细胞比例增加(63.18%±5.21%vs52.81%±4.36%,P<0.05;36.49%±3.93%vs26.70%±2.62%,P<0.05),G2期细胞减少(0.33%±0.02%vs20.49%±3.71%,P<0.05),克隆形成率明显高于亲本细胞(69%vs24%,P<0.05),对罗丹明的摄入减少,排出增多.结论:成功建立了人食管癌顺铂耐药细胞系Ec9706/cDDP,且耐药性较稳定.

AIM： To establish a cisplatin-induced human esophageal carcinoma drug-resistant cell line Ec9706/cDDP and study its biological characteristics. METHODS： The drug-resistant cell line Ec9706/ cDDP was established in culture by exposing Ec9706 parent cells to moderate concentration of cisplatin （cDDP） over a period of 9 months. The resistance index to cDDP was determined by the methyl thiazolyl tetrazolium （MTT） assay. Cell growth curve was painted and the doubling time was accounted. Flow cytometry （FCM） was performed to determine cell cycle. Fluorescence activated cell analysis （FACS） was employed to determine the concentration of fluorescence dye of rhodamine 123 （Rh123） in the cells for the evaluation of drug-absorptive and expelling capacity. RESULTS： The resistant index of Ec9706/cDDP was 15.7. When Ec9706/cDDP was stored with cDDP at -196℃ for 3 months and then back into the normal environment, its characteristic of anti-tumor drug resistance was still maintained. But after being cultured in RPMI-1640 without cDDP for a period of 1 month, Ec9706/cDDP showed a decrease in the resistant index. As compared with the parent cells, Ec9706/cDDP exhibited a prolonged doubling time （29.79±0.48 h vs 25.79±0.45 h, P 〈 0.05） and lower growth rate. The percentages of cells in S and G0/G1 phase were significantly increased in Ec9706/cDDP in comparison with those in Ec9706 （36.49% ± 3.93% vs 26.70% ± 2.62%, P 〈 0.05; 63.18% ± 5.21% vs 52.81% ± 4.36%, P 〈 0.05） while the percentage of G2-phase cells was decreased （0.33% ± 0.02% vs 20.49% ± 3.71%, P 〈 0.05）. In soft agar growth test, Ec9706/cDDP cells formed more cell clones than Ec9706 cells （69% vs 24%, P 〈 0.05）. Finally, the concentration of Rh123 in Ec9706/cDDP was lower than that in Ec9706. CONCLUSION： The newly established stable cell line Ec9706/cDDP possesses typical multidrug resistant characteristics and its drug-resistant phenotype is stable.