逆转录病毒介导的GM-CSF在骨髓基质细胞中基因转移与表达

为了探索逆转录病毒介导的GM-CSF在骨髓基质细胞(BMSC)中的基因转移与表达,采用电穿孔法将重组质粒pLXSN/GM和空载体pLXSN转染PA317细胞,经G418筛选,用抗性克隆培养上清液成功地感染了BMSC。经PCR和Southern blot分析证实BMSC基因组中整合有外源NeoR和GM-CSF cDNA,原位杂交显示有较强的GM-CSFmRNA的表达,经增殖法和集落形成法表明转染的BMSC分泌较多的GM-CSF。据此表明,转染的BMSC较未转染BMSC具有更强的支持造血功能,提示BMSC可作为造血功能障碍基因治疗的靶细胞,为GM-CSF基因治疗血液病奠定基础。

Study on Retrovirus-Mediated Murine GM-CSF cDNA Transfer and Expression in Bone Marrow Stromal Cells

In order to investigate the feasibility of retrovirus-mediated GM-CSF gene transfer into bone marrow stromal cells (BMSC), granulocyte/macrophage colony-stimulating factor (GM-CSF) cDNA was recombined with retroviral vector pLXSN. After this recombinant plasmid was transfected into retro-virus packaging cell line PA317 by eletroporation,. G418 resistant clones could produce GM-CSF cDNA recombinant retrovirus successfully which could infect stromal target cells and make them form typical resistant clones in G418 selective medium. In the genome of these infected target cells, NeoR gene and GM-CSF cDNA were successfully integrated, the GM-CSF mRNA expression in the BMSC was detected by in situ hybridization and the specific activity of GM-CSF in the cultural medium was assessed. These results demonstrated that BMSC transfected with GM-CSF cDNA could significantly augment GM-CSF in vitro and might become target cells of gene therapy, and provided a basis for further investigation on hematopoietic reconstitution of BMSC mediated GM-CSF gene therapy.