mdr-1和DHFR双基因共转染人CD34+细胞拓宽造血细胞耐药谱的体外研究

目的　探讨将多药耐药基因 (mdr 1)和二氢叶酸还原酶基因 (DHFR)同时导入人CD3 4 细胞 ,以拓宽造血细胞耐药谱 ,改善骨髓耐受联合化疗的可行性。方法　将以造血细胞中高表达的逆转录病毒载体FMCF为基本结构骨架 ,通过引入IRES序列构建获得含mdr 1和DHFR(L2 2Y)双耐药基因的逆转录病毒载体pSF DIM ,通过脂质体介导包装 ,单嗜性和双嗜性包装细胞上清交叉感染提高病毒滴度。低温离心病毒上清转染人脐血CD3 4 细胞 ,用流式细胞仪检测P gp的表达 ,基因组PCR检测外源性耐药基因的整合 ,CFU GM培养观察耐药性变化。结果　逆转录病毒载体pSF DIM转导人脐血CD3 4 细胞后 ,P gp的表达较未转基因组增加了 10 .98% ;基因组PCR同时检测到两种外源性耐药基因的整合 ;与未转基因组比较 ,在 48nmol/L甲氨蝶呤和 10ng/ml及 12ng/ml紫杉醇 (商品名Taxol)浓度水平 ,CFU GM集落形成显著增加 (P <0 .0 5 )。结论　重组双耐药基因逆转录病毒载体pSF DIM可以有效介导mdr 1和DHFR双耐药基因进入人脐血CD3 4 细胞并且获得共表达 ,拓宽了造血细胞耐药谱

In vitro cotransfer human multidrug resistance gene(mdr-1) and dihydrofolate reductase gene(DHFR) into human CD34+ progenitor cells to broaden the spectrum of drug resistance

Objective To explore the feasibility of cotransferring human mdr 1 gene and DHFR gene into human CD 34 progenitor cells to broaden the spectrum of drug resistance and improve the tolerance of myelosuppression following combination chemotherapy. Methods The recombinant retroviral vector pSF DIM containing mdr 1 and DHFR (L22Y) gene was constructed by introducing IRES sequence into vector FMCF which enable highly efficient gene expression in early hematopoietic cells. The retrovirus titers were raised by repeated supernatant cross infection between the amphotropic and ectropic retroviral packaging cells. Human CD 34 progenitor cells were transduced by supernatant infection. Expression of P gp was detected by flow cytometry. Integration of the foreign drug resistance gene in CD 34 cells was determined by PCR. Drug resistance was evaluated by CFU GM assay. Result Integration of the two foreign drug resistance genes was detected in the CD 34 cells after pSF DIM transduction. Compared with the untransduced group, the expression of P gp elevated by 10.98% after gene transduction and the CFU GM yields were significantly increased at 48 nmol/L of MTX and 10?ng/ml or 12 ng/ml of taxol(P<0.05). Conclusion The retroviral vector pSF DIM can mediate mdr 1 and DHFR gene integration and co expression in human hematopoietic progenitor cells so as to broaden the spectrum of drug resistance.