应用IRES序列研究人FL与Tpo基因在转染HFCL细胞中的表达

目的　探讨应用内部核糖体进入位点（ＩＲＥＳ）序列对逆转录病毒介导的人Ｆｌｔ３ 配基（ＦＬ）与血小板生成素（Ｔｐｏ） 基因在骨髓基质细胞系ＨＦＣＬ中的表达。方法　利用基因重组技术将ＩＲＥＳ序列与ＦＬ和Ｔｐｏ 基因构建到逆转录病毒载体中，脂质体法将重组质粒ｐＬＦＴＳＮ 和空载体ｐＬＸＳＮ转染ＰＡ３１７细胞，经Ｇ４１８ 筛选。用抗性克隆培养上清感染ＨＦＣＬ细胞，ＲＴＰＣＲ和基因组ＤＮＡＰＣＲ分析ｍＲＮＡ的表达及基因组整合情况，ＣＦＵＧＭ 集落法和Ｔｐｏ 依赖株法测定生物学活性。结果　成功构建出含ＩＲＥＳ序列的ＦＬ与Ｔｐｏ 基因逆转录病毒载体，ｍＲＮＡ水平及基因组中整合有ＦＬ与Ｔｐｏ 基因，生物学活性测定表明转染的骨髓基质细胞同时分泌ＦＬ与Ｔｐｏ。结论　ＩＲＥＳ序列调控ＦＬ与Ｔｐｏ 双基因在骨髓基质细胞中同时独立表达，为进一步研究转基因骨髓基质细胞对造血调控的影响奠定了基础。

Expression of human flt3 ligand and thrombopoietin genes in a bone marrow stromal cell line by internal ribosome entry site (IRES)sequence

Objective To explore the feasibility of retroviral mediated Flt 3 ligand (FL) and thrombopoietin (Tpo) genes transferred into and expressed in a bone marrow stromal cell line HFCL by internal ribosome entry site(IRES)sequence. Methods IRES sequence, FL and Tpo cDNA were recombined with retroviral vector pLXSN by gene recombination technology. The recombinant plasmid was transferred into retrovirus packaging cell line PA317 by lipofectamine, and the resistant clones were selected by G418 selective medium. mRNA expression in HFCL cells and integration of genome DNA were assayed by RT PCR and genomic DNA PCR. The biological activities of FL and Tpo in the culture were investigated by CFU GM assay and Tpo dependent cell line TD 3,respectively. Results The recombinant plasmid pLFTSN was successfully constructed. In the genome of these transfected target cells, Neo gene and FL and Tpo cDNA were intergrated, the expression of FL and Tpo mRNA was detected in HFCL cells. The specific activities of FL and Tpo in the culture indicated that HFCL cells transfected with FL and Tpo cDNA could significantly express FL and Tpo in vitro. Conclusion FL gene and Tpo gene were simultaneously expressed in bone marrow stromal cell line by the regulation of IRES sequence. These results provide a basis for studies on hematopoietic regulation by gene transfected bone marrow stromal cells.