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Effects of erythropoietin gene transfer into skeleton muscle on renal anemia
Author(s): 
Pages: 2331-2334
Year: Issue:  20
Journal: THE JOURNAL OF PRACTICAL MEDICINE

Keyword:  贫血肾功能衰竭慢性红细胞生成素重组四环素调控系统基因治疗;
Abstract: 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pTRE2hyyg-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.
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