构建促红细胞生成素表达载体及其在肾小管上皮细胞中的表达

Construction and expression of a pcDNA3.1(-)-hEPO vector in human proximal tubular epithelial cells

目的构建促红细胞生成素(EPO)表达质粒,并在人近端肾小管上皮细胞中稳定表达。方法应用标准分子克隆技术构建EPO真核表达载体pcDNA3.1(-)-hEPO。磷酸钙法转染人肾小管近端上皮细胞(HK-2)。酶联免疫吸附法(ELISA)检测培养液上清中的EPO含量。MTT法绘制各组细胞生长曲线。结果构建获得EPO基因的表达质粒,并成功转染肾小管上皮细胞,后者经G418筛选,稳定表达EPO。转染后第21天,其表达量为同期转染的293细胞的(35.0±5.3)%。转染后HK-2细胞生长曲线未见明显改变。结论人近端肾小管上皮细胞可以稳定表达EPO,这为EPO基因治疗选择新靶点奠定了基础。

Objective To stably express human erythropoietin （EPO） gene in human proximal tubular epithelial cells （HK-2）. Methods Eukaryotic expression vector for human EPO gene was constructed by the standard molecular cloning method. The recombinant pcDNA3.1 （-）-hEPO vector was transfected to 293 cells and HK-2 cells by the calcium phosphate coprecipitation method. EPO in cultured supernatant was detected by ELISA. Proliferation of HK-2 cells was assayed by MTT method. Results After successful construction of the recombinant plasmid and transfection of the plasmid to HK-2 cells, EPO was temporarily expressed in transfected HK-2 cells, and was constantly expressed in ttansfected HK-2 after selection by G418. The expression level of EPO in transfectcd HK- 2 cells was （35.0±5.3）% of that in transfected 293 cells. HK-2 cells stably expressing transfected human EPO did not show any changes of cell growth. Conclusion Human EPO cDNA can be stably expressed in human proximal tubular epithelial cells （HK-2）, suggesting a promising approach for EPO gene therapy.