新型逆转录病毒载体介导的人多药耐药基因转移小鼠造血干／祖细胞

为探讨逆转录病毒介导的外源基因转导对骨髓造血重建的影响,我们以人多药耐药基因(mdr-1)为报告基因,采用包含Friend脾灶形成病毒(spleen focus-forming vims)和鼠胚胎干细胞病毒序列的新型逆转录病毒载体SF-MDR,以病毒包装细胞与小鼠造血细胞体外共培养法进行基因转染,并对基因转导后造血细胞的体外耐药能力及体内造血重建能力进行了观测。结果表明,该载体可有效地介导mdr-1基因转导,明显提高小鼠骨髓造血细胞对秋水仙素及紫杉醇的耐受能力,对细胞体内造血重建能力没有影响,体内移植8个月后经紫杉醇体内筛选,7/7的小鼠可于外周血细胞基因组DNA中检出外源mdr-1基因的存在。

A Novel Retroviral Vector-Mediated Human Multidrug Resistance Gene mdr-1 Transfer into Murine Bone Marrow Hematopoietic Stem/Progenitor Cells

In order to investigate the effect of gene transfer on bone marrow cell reconstitution potential, a novel retro-viral vector SF-MDR was adopted. The vector was constituted by mdr-1 (reporter gene), the sequences of Friend's spleen focus-forming virus and murine embryonic stem cell virus. The mdr-1 gene transduction on murine hematopoietic cells was performed by in vitro co-culture of viral packaging cells with murine marrow cells. The resistance capability to colchicine and taxol was determined by CFU-GM assay in vitro and the reconstitution potential of hematopoiesis was observed in vivo. The results demonstrated that this vector could efficiently mediated the mdr-1 transduction, and the mdr-1 gene modified marrow cells showed high proportion of resistance to both colchicine and taxol. There was no any change on the hematopoietic reconstitution potential of murine marrow cells in vivo. After 8 months of transplantation with mdr-1 gene transferred-marrow cells and treated with taxol, the mdr-1 positive cells were detected from genomic DNA of peripheral blood cells in all of the 7 experimental mice by PCR.