四环素调控促红细胞生成素基因转导骨骼肌细胞治疗肾性贫血的研究

Effects of erythropoietin gene transfer into skeleton muscle on renal anemia

目的:克隆、构建、鉴定重组人促红细胞生成素(rhEPO)基因四环素调控真核表达载体;探讨四环素系统对rhEPO基因表达的调控。方法:以含rhEPO全长cDNA的CHO细胞为模板,通过RT-PCR方法扩增出rhEPO基因,将其克隆到真核表达载体pTRE2hyg的多克隆位点,构建成pTRE2hyg-EPO,进行酶切及测序鉴定。将pTRE2hyg-EPO及Tet-On质粒转导入SD大鼠骨骼肌细胞,用RT-PCR、Western印迹实验和半固体细胞培养实验检测rhEPO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:(1)成功构建了四环素调控真核表达质粒pTRE2hyg-EPO,经测序鉴定与所需EPO基因序列一致。(2)rhEPO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。生成的EPO具有生物活性,可明显刺激骨髓红系细胞的生长。结论:在细胞水平四环素系统可控制转导的EPO基因的表达,并且生成有生物活性的EPO,达到基因治疗的要求。

Objective To clone, construct and identify a tetracycline response eukaryotic expressing plasmid encoding recombination human erythropoietin （rhEPO） gene and to explore the regulation of the tetracycline gene expression system on the rhEPO gene expression. Methods Amplified the rhEPO gene by RT-PCR from CHO cells which contain the total length eDNA of rhEPO gene, PCR products were cloned into eukaryotic expressing plasmid pTRE2hyg, sequenced and done restriction endonuclease disgesdon analysis. Transfer pTRE2hyg-EPO and Tet-On plasmid were transferred into rat skeletal muscle cell. The rhEPO gene expression and its biological activity were measured by RT-PCR, Western blot and semisolid colony culture experiment, and the regulation of the tetracycline gene expression system on rhEPO gene expression were observed. Results （1） pTRE2hyg-EPO eukarytie recombinant had been constructed sueeessfuUy, and kept the same sequences with source rhEPO gene. （2） rhEPO was expressed in rat skeletal muscle cell and regulated by the tetracycline gene expression system. Producted erythropoietin had biological activity and could obviously stimulate bone marrow cells growth. Conclusion The tetracycline gene expression system can regulate the pTRE2hyg-EPO gene expression at cell level. Produced erythropoietin have biological activity, and meet the requirement of gene therapy.