人舌癌细胞耐药系Tca8113/BLM的建立及其耐药机理的分析

目的 :建立舌癌平阳霉素 (BleomycinA5Hydrochloride ,BLM)耐药细胞系Tca8113/BLM ;研究Tca8113/BLM细胞P 糖蛋白 (P glycoprotein ,P gp)和多药耐药相关蛋白 (MRP)表达变化与其耐药的相关性。方法 :采用大剂量BLM(30 μg/ml)反复间歇 2 4小时暴露法处理舌癌细胞系Tca8113细胞 ;用MTT法检测该耐药细胞模型的多药耐药性 ;激光共聚焦显微镜和流式细胞仪检测P gp、MRP ;并用流式细胞仪检测细胞周期的分布 ,细胞内阿霉素 (ADM)蓄积 ,以及经典钙离子生物泵阻断剂逆转剂黄体酮 (Progesterone,Prog)对ADM蓄积的影响。结果 :①Tca8113/BLM耐药性稳定 ,耐药指数为 12 ,且对其它化疗药物产生交叉耐药性 ;②Tca8113/BLM的倍增时间延长 ,G1期细胞减少 ,G2和S期细胞增多 ;③P gp和MRP在Tca8113/BLM细胞中强阳性表达 ,在Tca8113细胞中弱阳性表达 ,两者相比差异有显著性 (P <0 .0 1) ;④ADM在Tca8113/BLM细胞内蓄积明显低于Tca8113细胞 (P <0 .0 5 ) ;⑤Prog能显著提高Tca8113/BLM细胞内ADM蓄积 ,而对Tca8113细胞内ADM蓄积无明显作用。结论 :建立了舌癌BLM耐药细胞系Tca8113/BLM ,发现Tca8113/BLM的耐药性与P gp和MRP过表达有密切关系

Establishment of human tongue cancer cell line Tca8113/BLM to drug resistance and analysis of mechanism of its drug resistance

Purpose:In order to establish a human tongue cancer cell line resistent to bleomycin(BLM) and to study the relationship between P-gp and MRP expression and drug-resistance. Methods:Human tongue cancer cell line Tca8113 cells were repeatedly treated with high dose of BLM (30(g/ml)to establish the drug-resistant cell line Tca8113/BLM. The expression of P-gp and MRP in Tca8113/BLM and its parental counterpart Tca8113 was analyzed by laser confocus microscope(LCSM) and flow cytometry(FCM). The cell cycle distribution, ADM congregation and effect of reversing agent Progesterone on multidrug resistance(MDR) in both cell lines were detected by FCM. Results:Resistance of Tca8113/BLM cell line to BLM was stable, and its drug-resistance index is 12. It also produced resistance to other chemotherapy drugs. Compared with Tca8113, the doubling time of Tca8113/BLM was prolonged, G_1 phase cell number decreased and S and G_2 phases cell number increased. LCSM observation showed that expression levels of P-gp and MRP in Tca8113/BLM cells were significantly higher than those in Tca8113 cells (P<0.01). FCM analysis showed that intracellular concentration of ADM decreased evidently in Tca8113/BLM cells compared with Tca8113 cells, and Progesterone could increase the intracellular concentration of ADM in Tca8113/BLM cells, but progesterone had no significant effect on the intracellular concentration of ADM in Tca8113 cells. Conclusions:A human tongue cancer cell line resistant to BLM was established. Drug-resistance of Tca8113/BLM cells is associated with over-expression of P-gp and MRP. Tca8113/BLM cell line provides an ideal model for MDR research.