逆转录病毒介导GM-CSF在造血祖细胞中的基因转移

Study on the Retrovirus Mediated Transfer of GM-CSF cDNA in Hematopoietic Progenitor Cells

为了探索GM-CSF基因治疗的可行性,采用逆转录病毒载体介导的基因转移方法将小鼠GM-CSF cDNA逆转录病毒pLXSN/GM转染病毒包装细胞PA317,然后感染富含造血干、祖细胞群体,经含G418的体外半固体造血祖细胞培养,表现出较多的G418抗性CFU-GM生成,用PCR和Southem Blot方法分别检测出转化细胞基因组中整合有Neo^R基因和GM-CSF cDNA,原位杂交显示转化细胞有较强的GM-CSF mRNA表达,在Dexter培养中,GM-CSF修饰组的成熟非粘附细胞计数明显高于对照组(P<0.05).上述结果表明GM-CSF重组逆转录病毒成功地转入造血祖细胞并获得了表达,为下一步体内基因治疗奠定基础.

In order to investigate the feasibility of granulocyte-macrophage colony stimulating factor (GM-CSF) gene therapy, murine GM-CSF cDNA recombinant retrovirus pLXSN/GM was transfered into retrovirus-packaging cell line PA3 17 by electroporation, and the transfected cells were used to infect hematopoietic progenitor cells rich populations. Transfective efficiency of NeoR gene was detected by G418 resistant CFU-GM test, and the results showed 36% them. In the genome of the infected target cells, integrated NeoR gene and GM-CSF cDNA were identified successfully by PCR and Southern Blot analysis respectively. The recombinant plasmids were showed to be capable of expressing GM-CSF mRNA in hematopoietic cells by in situ hybridization. In Dexter culture system, the present of GM-CSF-producing transduced cells inscreased mature nonadherent cell numbers as compared to controls. These results demonstrated that recombinant plasmids were successfully transfected into hematopoietic progenitor cells, and expressed in the cells. Therefore, it provided a basis for further investigation of gene therapy.