Transfection of Multidrug Resistant Gene into Human Hematopoietic Stem Cells and its Clinical Application in Bone Marrow Transplantation

多药耐药基因（MDR1）编码一种膜结合p糖蛋白（分子量约为170 kDa），在结构无关的细胞毒性药物如长春花生物碱、蒽环类药物、秋水仙碱、放线菌素D等药物的外排转运中起重要作用，其中一些药物目前已被临床用于癌症化疗。最近，MDR1基因的分离为在分子水平上阐明癌细胞的多药耐药带来了希望。我们的目标是将MDR1基因引入人造血干细胞，以获得具有多药耐药表型的正常血细胞。从单核细胞白血病细胞系NOMO1/ADR cDNA文库中克隆出MDR1 cDNA片段，全长4.3 kb。该cDNA已插入逆转录病毒载体pCO1中。将该载体转染到包装细胞系PA-12中产生病毒。该病毒赋予犬肾细胞系MDCK多重耐药表型。通过单克隆抗体的筛选和分选方案，从正常骨髓中获得了20 - 30个浓度的人多能干细胞。将含有MDR1 cDNA的病毒感染这些细胞。用含药培养基培养的菌落数量没有增加。观察存活菌落中活细胞的增加。初步分析了新表达系统的潜在用途。在产生细胞中完成了病毒滴度的50次扩增。然而，也发现了病毒基因组的大量缺失，这表明基因缺陷可能是由快速基因复制引起的。

Multidrug Resistant gene (MDR1) encodes a membrane-binding P-glycoprotein (approximately 170 kDa molecular mass) that plays an important role in efflux transportation of structurally unrelated cytotoxic drugs such as Vinca alkaloids, anthracycline, colchicine, actinomycin D and some other drugs, some of which are now clinically used in cancer chemotherapy.Recently, isolation of the gene for MDR1 has raised hope that multidrug resistance of cancer cells might be elucidated at a molecular level.We aimed to introduce MDR1 gene into human hematopoietic stem cells to obtain otherwise normal blood cells with a multidrug resistant phenotype.MDR1 cDNA fragment (4.3 kb in length) was cloned from cDNA library derived from monoblastic leukemia cell line, NOMO1/ADR. This cDNA has been inserted into a retroviral vector pCO1. Virus was produced after transfection of this vector into a packaging cell line PA-12. This virus conferred multidrug resistant phenotype on dog kidney cell line MDCK.Twenty to 30-hold concentration of human multipotent stem cells from normal bone marrow was achieved by panning and sorting protocol with monoclonal antibodies. Virus containing MDR1 cDNA was infected into these cells. There is no increase in the number of colonies cultured with drug-containing medium. Increment of viable cells in surviving colonies was observed.We preliminarily analyzed a potential use of new expression system. Fifty-hold amplification of virus titer was accomplished in the producer cells. However, a large deletion of viral genome was also identified, suggesting that the gene defect might be caused by a rapid gene duplication.