重组腺病毒Ad-EGFP/HIF-1α转染大鼠内皮祖细胞

Transfection of recombinant adenovirus Ad-EGFP/HIF-1α to rat endothelial progenitor cells in vitro

目的:研究含缺氧诱导因子(HIF-1α)及增强型绿色荧光蛋白(EGFP)的重组腺病毒(Ad-EGFP/HIF-1α)感染大鼠内皮祖细胞(EPCs)的可行性.方法:采用percoll密度梯度离心法分离SD大鼠骨髓单个核细胞,VEGF,bFGF,EGF诱导培养,观察其形态学变化,免疫细胞化学染色鉴定其表面抗原.Ad-EGFP/HIF-1α在HNK293细胞中进行扩增,之后转染体外培养的EPCs,分别于24,48,72,96h采用倒置荧光显微镜下观察细胞表达绿色荧光蛋白(GFP)情况.MTT检测细胞生长活性;流式细胞术检测转染率并用RT-PCR法测定HIF-1α在EPCs中的表达.结果:倒置荧光显微镜下可观察到大量绿色荧光,随着时间的延长,绿色荧光逐渐增多.MTT法检测细胞在24,48,72,96h的存活率分别为99.83%,99.70%,99.32%,99.57%;流式细胞仪计数随时间的延长,转染率逐渐升高,24,48,72,96h的转染率分别为23.05%,45.94%,78.91%,85.64%.RT-PCR可检测到被感染细胞HIF-1α的表达.结论:重组腺病毒Ad-EGFP/HIF-1α能够有效地感染EPCs,转染后在mRNA水平可检测到EPCs中有HIF-1α的表达,并对EPCs活性无明显影响,为进一步研究转基因的EPCs移植促血管新生奠定基础.

AIM：To investigate the feasibility of transfection of recombinant adenovirus Ad-EGFP/HIF-1α to rat endothelial progenitor cells（EPCs）.METHODS：The mononuclear cells were isolatedfrom rat bone marrow using percoll density gradient centrifugation,then induced with VEGF,bFGF and EGF.The expression of cell markers was assessed by immunocytochemistry.Ad-EGFP/HIF-1α was amplified in HNK293 cells,and then transfected EPCs in vitro.Expression of enhanced green fluorescent protein（EGFP）in infected EPCs was observed by fluorescence microscope,cell growth activity was detected by MTT,transfection efficiency was detected by flow cytometry,and expression of HIF-1α in EPCs was verified by RT-PCR analysis.RESULTS：After EPCs were infected with recombinant adenovirus,green fluorescence was found and increased gradually with the time in EPCs.The survival rates of EPCs had no significant change after transfection,and the transfection rate increased gradually with the time.Hypoxia inducible factor 1α（HIF-1α）was detected in infected cells by RT-PCR.CONCLUSION：The recombinant adenovirus Ad-EGFP/HIF-1α can infect EPCs effectively,without significant effect on activity of EPCs.