【摘要】 目的:将人低氧诱导因子1-α(HIF1-α)转染人骨髓内皮祖细胞(EPCs),为探讨转染HIF1-α基因的EPCs对梗塞心肌血管修复及血管再生能力提供依据。方法:密度梯度法分离人骨髓中单个核细胞,以1×106.mL-1细胞浓度接种于用纤维连接蛋白包被的培养皿中,培养14d后采用RT-PCR法检测内皮细胞特异性成分ecNOS、flk-1的表达;采用acLDL-Dil和FITC-UEA-1对细胞染色;采用LipofectamineTM 2000介导PCDNA3.0-HIF1-α质粒转染EPCs,RT-PCR检测HIF1-α的转录;ELISA检测细胞上清中VEGF表达;免疫印迹法检测HIF1-α蛋白表达。结果:培养第5天起,部分圆形单核细胞转化为梭形贴壁EPCs,7d左右出现由数十个细胞形成的细胞集落,10d后形成明显克隆。RT-PCR检测ecNOS在548bp处出现条带,flk-1在819bp处出现条带;acLDL-Dil和FITC-UEA-1双染色阳性;RT-PCR在383bp处出现特异性条带;ELISA转染HIF1-α的EPCs上清中VEGF含量为(20.53±2.33)μg·L-1,未转染HIF1-α的EPCs上清中VEGF含量为(3.96±1.67)μg·L-1,两组比较差异有显著性(P<0.05)。免疫印迹法(Western blotting)检测在相对分子质量120000处出现条带。结论:PCDNA3.0-HIF1-α质粒成功转染EPCs。更多还原

【Abstract】 Objective To transfect PCDNA3.0-HIF1-α into the endothelial progenitor cells(EPCs),in order to provide the foundation for exploring myocardial vascular repairing capacity and angiogenesis of the EPCs transfected with HIF gene.Methods The EPCs were separated by the method of density gradient separation in the human bone marrow,and then the cells were inoculated in the fibronectin-packaged petri dishes complete medium with the concentration of 1×106·mL-1,and cultivated for 14 d. The expressions of the endothelial cell-specific components ecNOS and flk-1 were detected with RT-PCR; the cells were stained with acLDL-Dil and FITC-UEA-1; the PCDNA3.0-HIF1-α plasmid was transfected into the EPCs mediated by LipofectamineTM 2000,the transcription of HIF1-α was determined with RT-PCR; the expression of VEGF was detected with enzyme-linked immunosorbent assay (ELISA); the expression of HIF1-α was detected with Western blotting.Results After cultivated for 5 d,some round monocytes transformated into spindled inherent EPCs;7 d later,there were some cell colonies containing dozens of cells;10 d later,there were obvious cell clones; RT-PCR showed ecNOS band in 548 bp,flk-1 band in 819 bp; acLDL-Dil and FITC-UEA-1 double-staining was positive; RT-PCR showed specific band in 383 bp; ELISA showed the content of VEGF in the supernatant of EPCs transfected with HIF1-α was (20.53±2.33) μg·L-1,and it was (3.96±1.67) μg·L-1 in the EPCs transfected without HIF1-α,there was significant difference between two groups (P<0.05).Western blotting showed specific band in 120 000.Conclusion PCDNA3.0-HIF1-α is successfully transfected into EPCs.更多还原