FLT3配基在人骨髓基质细胞系中的基因转移与表达

THE TRANSFECTION AND EXPRESSION OF RETRO VIRAL-MEDIATED HUMAN FL cDNA IN BONE MARROW STROMAL CELL LINE

目的 :研究逆转录病毒介导的FL在骨髓基质细胞系HFCL中的表达。方法 :采用脂质体法将重组质粒pLF SN/HFCL和空载体 pLXSN/HFCL转染包装细胞PA317,G418筛选抗性克隆 ,用抗性克隆上清液感染HFCL。RT PCR和基因组DNA PCR检测外源基因mRNA水平的表达及染色体的整合 ,小鼠CFU GM集落法检测FL生物学活性。结果 :在mRNA水平有FL的表达 ,染色体基因组中整合有标记neo基因和FL基因。活性测试结果显示转染的骨髓基质细胞分泌FL。结论

Aim: The expression of retroviral mediated FL gene transfer into bone marrow stromal cell line HFCL was studied. Methods: FLT3 ligand (FL) cDNA was recombined with retroviral vector pLXSN by gene recombination technology. The recombinant plasmid was transferred into retrovirus packaging cell line PA3 17 by lipofectamine, and the resistant clones were selected by G418 selective medium. The mRNA expression in HFCL cells and integration of genome DNA were assayed by RT PCR and genomic DNA PCR. The biological activity of FL in the culture was investigated by mouse bone marrow CFU GM assay. Results: The recombinant plasmid pLFSN was successfully constructed. The expression of FL mRNA was detected in HFCL cells.In the genome of these infected target cells, neo gene and FL cDNA were successfully expressed.The biological activity of FL in the culture demonstrated that HFCL cells transfected with FL could significantly augment FL in vitro . Conclusion: These results suggest that bone marrow stromal cell lines might become target cells of gene therapy.