转染VEGF基因对内皮祖细胞生长的影响研究

Experimental study of rat endothelial progenitor cells transfected with VEGF 165 gene

目的探讨血管内皮生长因子(vascular endothelial growth factor,VEGF)基因转染大鼠内皮祖细胞的方法,分析转染VEGF基因对内皮祖细胞生长的影响。方法分离大鼠骨髓内皮祖细胞并诱导其分化,采取FITCUEA-I和Dil-Ac-LDL双染色法验证;采用腺病毒包装VEGF165基因,按照不同比例转染内皮祖细胞后,通过荧光显微镜观察质粒自带绿色荧光蛋白验证转染效率;以未转染及转染空载体为对照;采用MTT法验证转染VEGF165基因对内皮祖细胞增殖的影响;采用ELISA法检测转染后细胞培养上清中的VEGF含量。结果分离诱导的大鼠内皮祖细胞分化良好,FITC-UEA-I及Dil-Ac-LDL均呈阳性;按照细胞病毒比1:50转染内皮祖细胞的转染效率最高,且细胞状态不受影响;转染VEGF165基因的内皮祖细胞能分泌大量的VEGF,提高培养上清液中的总VEGF水平,其增殖能力也明显强于空质粒转染组和未转染组。结论通过腺病毒载体转染VEGF165基因可以促进内皮祖细胞分泌VEGF,提高内皮祖细胞的增殖能力。

Objective To investigate the methodology of transfecting vascular endothelial growth factor（VEGF）gene to endothelial progenitor cells and detect the effect of VEGF transfection to endothelial progenitor cells.MethodsMononuclear cells from rat bone marrow were separated and induced for differentiation,and double staining of FITCUEA-I and Dil-Ac-LDL was used to characterize the induced endothelial progenitor cells.VEGF165 gene was incorporated into adenovirus and transfected into endothelial progenitor cells according to different cell virus ratio,the green fluorescence protein was used as readout to detect the efficiency of transfection.Theproliferation of VEGF transfected endothelial progenitor cells was detected by MTT colorimetric assay,and the VEGF in the supernatant of culturing cells was detected by enzyme-linked immunosorbent assay（ELISA）.Results The endothelial progenitor cells were well induced and differentiated,both positive for FITC-UEA-I and Dil-Ac-LDL staining.Transfection using 1：50as cell virus ratio showed the fittest efficiency and survival rate.Transfected endothelial progenitor cells secreted VEGF and up-regulated the level of VEGF in the supernatant,and the cells transfected with VEGF showed superior capacity of proliferation than vector transfected cells or non-transfected cells.Conclusion VEGF165 gene transfected endothelial progenitor cells could self-secrete VEGF and thus showed superior capacity of proliferation.