四环素对EPO基因表达的调控研究

Regulation of erythropoietin expression by tetracycline regulatory system.

目的探讨四环素调控系统对重组人促红细胞生成素基因表达的调控。方法将pTRE2hyg-EPO及Tet—On质粒转导人SD大鼠骨骼肌细胞，用RT-PCR、Western印迹实验及红系集落生成实验检测hEPO基因在大鼠骨骼肌内的表达和蛋白的活性，并观察四环素系统对EPO表达的调控作用。结果重组人EPO基因可以在大鼠骨骼肌原代细胞中表达，并受四环素调控系统的控制，并与诱导药物剂量成正比。生成的促红细胞生成素具有生物活性，可明显刺激骨髓红系细胞的生长。结论在细胞水平四环素调控系统可控制转导的EPO基因的表达，并且生成有生物活性的促红细胞生成素，达到基因治疗的要求。

Objective To in vestigate the effect of regulating rhEPO gene expression by tetracycline gene expression regulating system. Method Rat primary skeletal muscle cells were cultured for gene delivery use. Rat primary skeletal muscle cells were engineered for doxycycline-inducible and skeletal muscle-specific expression of rhEPO cDNA by co-transfection with pTRE2hyg-EPO and Tet-ON plasmids and the regulating effect of tetracycline system on expression of EPO was observed. Results The transfection positive cells could secret EPO which showed biological activity and was regulated by tetracycline system. EPO expression could be iteratively switched on and off, depending on the presence or absence o doxycycline,and the amount of EPO secretion increased/decreased with the increase/decrease of dose of doxycycline. Conclusions Tetracycline gene expression system provides a suitable control system for regulating the expression of EPO gene with production of bioactive erythropoietin which meet the criterion of gene therapy.