膀胱肿瘤抗药机制的实验研究

The mechanism of drug resistance in bladder cancer

应用Ｌｉｐｏｆｅｃｔｉｎ介导的基因转移技术，将人ＭＤＲ１－ｃＤＮＡ导入ＥＪ膀胱癌细胞，经６０、１２０和２４０μｇ／Ｌ秋水仙素的逐级筛选，挑出两个耐药的克隆细胞系，分别培养在２４０μｇ／Ｌ或４８０μｇ／Ｌ秋水仙素的培养基中，命名为ＥＪ－Ｒ２４０或ＥＪ－Ｒ４８０。对照组ＥＪ膀胱癌细胞在６０μｇ／Ｌ秋水仙素的培养基中不能生存。Ｓｏｕｔｈｅｎ、ＲＮＡ斑点杂交分析和免疫组化分析显示，人ＭＤＲ＿１－ｃＤＮＡ已整合在转染细胞的基因组中，并有相应的ｍＲＮＡ和蛋白产物Ｐ－糖蛋白的表达。药敏试验结果显示ＥＪ－Ｒ２４０和ＥＪ－Ｒ４８０对秋水仙素的耐受性分别是对照组的７３．６和８４．２倍。资料表明，ＭＤＲ＿１基因及其产物Ｐ－糖蛋白的过度表达可赋予转染细胞的抗药表型。因此，本研究不仅从基因水平上证实ＭＤＲ＿１基因及Ｐ－糖蛋白的异常表达可能是膀胱癌耐药性产生的一个重要途径或机制，同时也为研究ＭＤＲ＿１基因介导的多项耐药以及寻找有效的逆转途径提供了一个理想的实验模型。

Human multidrug resistance(MDR1)gene was introduced into EJ bladder carcinoma cells by lipofectin-mediated gene transfer.The transfected cells were selected by stepwise increase of colchicine concentration(60,120 and 240μg/L).Finally,two drug resistant clones,growing stably in complete 1640 medium containing 240μg/L and 480μg/L colchcine,were obtained and were designated as EJ-R240 and EJ-R480.On the other hand,the control EJ cells could not live in the complete 1640 medium containing even 60μg/L colchicine.The southern, RNA dot hybridization and immunohistochemistry assay designated that human MDR1 cDNA was integraded into genome of the transfected cells.The drug sensitivity test indicated that resistance of EJ-R240 and EJ-R480 to colchicine has been 73.6 and 84.2 fold higher than the control parental cells. The result indicated that over expression of MDR1 gene and P -glycoprotein could confer the drug resistance on bladder carcinoma cells.It was claimed that such transfected cells might serve as an ideal model to study MDR1 mediated multidrug resistance and to search the effective means to reverse MDR1 gene mediated multidrug resistance.