PcDNA3.0-hVEGF165的构建及转染骨髓源性血管内皮祖细胞的体外研究

目的构建hVEGF165真核表达载体,进行血管内皮生长因子(VEGF)转染骨髓源性血管内皮祖细胞(EPC)的体外基础研究。方法设计引物后ECV304细胞克隆基因,构建真核表达型的PcDNA3.0-hVEGF165。EGM-2MV培养基培养大鼠骨髓中的单个核细胞,免疫细胞化学及电镜鉴定EPC,脂质体介导PcDNA3.0-hVEGF165转染EPC,转染后ELISA法检测培养上清中VEGF蛋白水平,MTT法评价VEGF转染EPC后对细胞活性的影响。结果构建真核表达型的PcDNA3.0-hVEGF165成功,EGM-2MV培养出的大鼠骨髓EPC能表达CD34、CD133、FLK-1等特征性的细胞表面标志,脂质体介导PcDNA3.0-hVEGF转染EPC能表达较高浓度的VEGF蛋白水平,VEGF转染后对EPC的增殖无影响。结论脂质体介导PcDNA3.0-hVEGF165转染EPC后能表达一定浓度的VEGF蛋白,并对EPC的活性无明显影响。

To construct a recombinant PcDNA3.0-hVEGF165 and to investigate the transfection on endothelial progenitor cell derived from bone marrow in vitro

Objective To construct a recombinant eukaryotic expression vector PcDNA3.0-hVEGF165 and investigate the basic study of transfection on endothelial progenitor cell derived from bone marrow in vitro. Methods Obtain the EPCS by culturing the mononuclear cell isolated from the rat's bone marrow in EGM-2MV. EPCS were identified by immunocytochemistry and electron microscope. PcDNA3.0-hVEGF165 transfect EPCs mediated by liposome. Detect the VEGF protein level in the cultural medium supernatant after VEGF transfection by ELISA. Evaluate the influence of EPCs' activity after transfection through MTT. Results We construct a recombinant eukaryotic expression vector PcDNA3.0-hVEGF165 successfully. It is a well method to enrich EPCS by EGM-2MV, and the EPCS obtained could express those distinctive cell surface markers such as CD34,CD133,FLK-1 and so on. EPCS could excrete a certain concentration of VEGF protein after PcDNA3.0-hVEGF transfection. No influence of EPCS proliferation could be found after transfection. Conclusion EPCS transfected by PcDNA3.0-hVEGF165 mediated by liposome could excrete a certain concentration of VEGF protein, and no influence of EPCS proliferation could be found after transfection.