Liu Yuanlin, Zhang Yi, Zhu Fuli, Tang Peixuan, Mao Ning
Institute of Basic Medical Sciences, Academy of Military Medical Sciences, Beijing 100850, China.
Zhonghua Xue Ye Xue Za Zhi. 2002 Feb;23(2):65-7.
To construct a retroviral-mediated vector of FLT3 ligand (FL) and express it in human bone marrow stromal cells.
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.
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.
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.
构建逆转录病毒介导的Flt3配体（FL）载体，并使其在人骨髓基质细胞中表达。
采用基因重组技术将FL cDNA插入逆转录病毒载体pLXIN。用脂质体将重组质粒pLFIN转入逆转录病毒包装细胞系PA317，经G418筛选阳性克隆。采用逆转录聚合酶链反应（RT-PCR）和基因组DNA-PCR检测人基质细胞中的mRNA表达及基因组DNA整合情况。用ELISA和小鼠骨髓CFU-GM试验研究培养物中FL蛋白的表达及其生物学活性。
成功构建重组质粒pLFIN。在这些转染的靶细胞基因组中，Neo基因和FL基因整合，转录出FL mRNA，表达的FL蛋白为4.35 ng/ml/24 h。培养物中FL的比活性表明，转染FL的人骨髓基质细胞在体外能显著表达FL。
逆转录病毒介导的FL基因在骨髓基质细胞中表达，且在转染细胞的上清液中可检测到FL的生物学活性。这些结果为基因转染骨髓基质细胞调控造血的研究提供了依据。
