FLT3配基基因在人骨髓基质细胞中的表达  

Expression of human FLT3 ligand gene in human bone marrow stromal cells

作　　者：刘元林[1] 张毅[1] 朱馥丽[1] 唐佩弦[1] 毛宁[1] 

机构地区：[1]军事医学科学院基础医学研究所,北京100850

出　　处：《中华血液学杂志》2002年第2期65-67,共3页Chinese Journal of Hematology

基　　金：北京市自然科学基金资助项目 ( 7992 0 2 7)

摘　　要：目的　构建人FLT3配基 (FL)逆转录病毒载体 ,并观察其在人骨髓基质细胞中的表达。方法　用基因重组技术 ,将FLcDNA插入逆转录病毒载体pLXIN ,获得重组载体pLFIN。采用脂质体法将重组质粒pLFIN转染包装细胞PA317,G418筛选抗性克隆 ,用抗性克隆上清液感染人骨髓基质细胞 ,RT PCR和基因组DNA PCR检测外源基因mRNA的转录及染色体的整合 ,ELISA法和小鼠CFU GM集落形成法检测FL蛋白表达量和生物学活性。结果　酶切鉴定表明成功构建重组逆转录病毒载体pLFIN ;染色体基因组中整合有外源基因FL和Neo;在mRNA和蛋白质水平均可检测到FL表达 ;2 4hFL蛋白表达量为 4.35ng ml,活性测试结果显示转染的骨髓基质细胞分泌FL。结论　逆转录病毒介导的FL在骨髓基质细胞中获得表达 ,并具有良好的生物学活性 。Objective To construct a retroviral-mediated vector of FLT3 ligand (FL) and express it in human bone marrow stromal cells. Methods FL cDNA was inserted into the retroviral vector pLXIN by gene recombination technology. The recombinant plasmid pLFIN was transferred into retrovirus packaging cell line PA317 by lipofectamine, and the positive clones were selected by G418. The mRNA expression in human stromal cells and integration of genome DNA were assayed by reverse transcriptase-polymerase chain reaction(RT-PCR) and genomic DNA-PCR. The expression of FL protein and its biological activities in the culture were investigated by ELISA and mouse bone marrow CFU-GM assay. Results The recombinant plasmid pLFIN was successfully constructed. In genome of these transfected target cells, Neo gene and FL gene were integrated, FL mRNA was transcripted and FL protein was expressed at 4.35 ng/ml /24 h. The specific activities of FL in the culture indicated that human bone marrow stromal cells transfected with FL could significantly express FL in vitro. Conclusion The retroviral-mediated FL gene was expressed in bone marrow stromal cells and the biological activities of FL were detectable in the supernatant of the transfected cells. These results provide a basis for studies on hematopoietic regulation by gene transfected bone marrow stromal cells.

关 键 词：FLT3配基 骨髓基质细胞 基因表达 

分 类 号：R346[医药卫生—基础医学]