逆转录酶病毒载体介导的人EPO基因在PA317细胞中的表达

Retroviral-mediated expression of erythropoietin gene in PA317 cells

本研究首先将人 EPO 基因构建到逆转录酶病毒载体 LXSN 中,经 DNA-磷酸钙共沉淀法,将重组载体转染到包装细胞 PA317中,转染的 PA317细胞在含 G418条件培养基中选择生长。通过 DNA-PCR 分析证明,重组逆转录酶病毒载体 LXSN-EPO 基因整合到 PA317细胞基因组 DNA 中。RT-PCR 和细胞原位杂交分析表明,EPO mRNA 在 PA317细胞中表达。转染 PA317细胞培养上清体外 EPO 生物活性测定显示,EPO 表达水平高达40 U/L,证明 EPO 基因在逆转录酶病毒载体的启动子控制下转录 EPO mRNA。我们的这一研究,为EPO 基因在靶细胞中表达的基因转移治疗肾性贫血提供了可能性。

Erythropoietin(Epo)is a glycoprotein regulating growth factor that functions to regulate erythropoiesis in the bone marrow. The majority of Epo is produced in renal peritubular cells in response to local oxy- genation.Decreased Epo production in chronic renal failure is overcome by repeat- ed injections of recombinant human Epo, thereby replacing transfusions and avoiding their iatrogenic complications.The cost of adequate replacement therapy is high,due in part to the short life span of the hormone in vivo.One alternative to rhEpo is to use the Epo gene as the pharmaceutical,either by transferring the human Epo gene to au- tologous cells in vitro and then transferring the modified cells back to the individual,or by directly transferring the Epo gene in vi- vo.In this study,we subclone human Epo gene into retroviral vector.Then recombi- nant retroviral vector was transfected into a packaging cell line PA317 by calcium phos- phate precipitation.The cells were cultured and selected by G418.DNA-PCR analysis demonstrated that the retroviral vector har- boring human Epo gene was integrated into PA317 cell genomic DNA.RT-PCR and in situ hybridization analysis showed that Epo mRNA was expressed in PA317 cells.The ex vivo bioāssay showed that the Epo ex- pression level is as high as 40 U/ml in the supernatant from culture of PA317 cells. This indicated that the Epo cDNA under control of retrovirus promoter translate Epo mRNA and the cells secrete functional Epo into medium.Our research provides the possibility of the expression of Epo gene in target cells for gene therapy.