逆转录病毒转染法建立耐药性大鼠CRBH-7919细胞系

目的:建立高效、稳定的大鼠CRBH-7919多药耐药细胞系.方法:利用逆转录病毒转染法将带有mdr1 cDNA全序列的逆转录病毒载体pHaMDR转入到大鼠CRBH-7919细胞中,MTT法检测细胞系在不同化疗药物作用下的存活率;免疫组化检测细胞的P-糖蛋白(P-GP)表达,RT-PCR检测细胞内mdr1 mRNA的表达量,PCR检测mdr1基因转移到细胞内的基因片段.结果:转基因的细胞系对阿霉素、丝裂霉素的耐药性分别提高 9和7.9倍,免疫组化见转基因细胞系P-GP表达增加,RT-PCR示细胞内mdr1 mRNA的表达量增加,PCR表明转基因细胞内扩增出mdr1片段.结论:利用逆转录病毒转染法成功建立了大鼠CRBH-7919多药耐药细胞系,该细胞系具有耐药强度高、耐药性稳定等特点.

Establishment of multidrug resistance rat CRBH-7919 cells line with retrovirus infection

Objective: To establish a multidrug resistance(MDR) rat CRBH 7919 cells line. Methods: Rat CRBH 7919 cells were transfected by mdr1 retrovirus supernatant and then named CRBH 7919 mdr1 cells. MDR of CRBH 7919 mdr1 cells were tested by MTT assay; expression of P GP on cell membrane and mdr1 mRNA in cells was detected by immunocytochemistry and RT PCR. The presence of mdr1 cDNA in genome of the cells was detected by PCR. Results: Compared with CRBH 7919 cells, the resistance of CRBH 7919 mdr1 cells was 9 times more to adriamycin and 7.9 times to mitomycin. CRBH 7919 mdr1 cells expressed higher P GP than CRBH 7919 cell membrane, they also showed much higher mdr1 mRNA level than CRBH 7919 cells. The PCR results confirmed that mdr1 cDNA had been transduced into the genome of the CRBH 7919 cells line. Conclusion: A MDR rat CRBH 7919 cells line is successfully established by gene transfer.