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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，ＲＴＰＣＲ和基因组ＤＮＡＰＣＲ分析ｍＲＮＡ的表达及基因组整合情况，ＣＦＵＧＭ 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。
Objective To investigate the expression of retrovirus-mediated human Flt3 ligand (FL) and thrombopoietin (Tpo) gene in bone marrow stromal cell line HFCL by using the internal ribosome entry site (IRES) sequence. Methods The IRES sequence and FL and Tpo genes were constructed into retroviral vectors by gene recombination technique. The recombinant plasmids pLFTSN and empty vector pLXSN were transfected into PA317 cells by lipofectamine and then screened by G418. HFCL cells were infected with the supernatant of the resistant clones. RT-PCR and genomic DNA were used to analyze the mRNA expression and genomic integration. The CFU-GM colonies and Tpo-dependent strains were used to determine the biological activity. Results FL and Tpo gene retroviral vectors containing IRES sequence were successfully constructed. The FL and Tpo genes were integrated into the genome and the biological activity of FL and Tpo genes were determined. The transfection of bone marrow stromal cells secreted FL and Tpo at the same time. Conclusions Both IR and Tpo double genes were independently expressed in bone marrow stromal cells at the same time, which laid the foundation for the further study of the effect of transgenic bone marrow stromal cells on hematopoietic regulation.