单分子成像显示视网膜血管内皮细胞膜上血管内皮生长因子受体2聚集状态

目的观察单个视网膜血管内皮细胞膜上血管内皮生长因子受体2(VEGFR2)的聚集状态。方法将猴视网膜血管内皮细胞(RF/6A)分为空白对照组(正常培养)、质粒转染组转染VEGFR2-绿色荧光蛋白(GFP)重组质粒]。采用共聚焦显微镜观察质粒转染组GFP的表达;实时荧光定量聚合酶链反应(qPCR)、蛋白免疫印迹法(Western blot)检测细胞中VEGFR2的mRNA和蛋白表达情况;单分子成像技术记录细胞膜上表达的单个VEGFR2-GFP分子的荧光强度分布和漂白步数,判断受体的寡聚或多聚状态。结果共聚焦显微镜观察发现,转染12 h后,质粒转染组细胞可见GFP绿色荧光。qPCR检测结果显示,质粒转染组细胞中VEGFR2、GFP mRNA表达较空白对照组显著升高,差异均有统计学意义(t=11.240、12.330,P<0.001、0.001)。Western blot检测结果显示,质粒转染组细胞VEGFR2蛋白表达较空白对照组增强,差异有统计学意义(t=8.346,P<0.01)。单分子成像结果显示,无配体刺激时RF/6A细胞膜表面上VEGFR2-GFP的荧光强度分布为双峰,其中单体、二聚体比例分别为86.0%、14.0%;通过计数GFP荧光漂白步数,静息状态下受体单体、二聚体、三聚体和四聚体比例分别为81.4%、12.9%、5.5%、0.3%。结论无配体状态下,VEGFR2在RF/6A细胞膜表面以单体和包括二聚体在内的多聚体形式共存,以单体为主。

Single-molecule imaging reveals stoichiometry of vascular endothelial growth factor receptor 2 in retinal vascular endothelial cells

Objective To observe the stoichiometry of vascular endothelial growth factor receptor 2(VEGFR2)on the retinal vascular endothelial cell membrane by single-molecule fluorescence imaging.Methods Rhesus monkey retinal vascular endothelial cells(RF/6A)were divided into blank control group(normal culture)and plasmid transfection grouptransfected with VEGFR2-green fluorescent protein(GFP)recombinant plasmid].The expression of GFP in the plasmid transfected group was observed by confocal microscope,and the expression of VEGFR2 in the cells was detected by real-time fluorescent quantitative polymerase chain reaction(qPCR)and Western blot.The fluorescence intensity distribution and bleaching steps of single VEGFR2-GFP molecule on the cell membrane were recorded by single-molecule imaging.The distribution of fluorescence intensity and the number of fluorescence bleaching steps of GFP were recorded.Results GFP green fluorescence was observed in the transfected cells 12 hours after transfection.qPCR results showed that the expression of VEGFR2 and GFP mRNA in the plasmid transfected group was significantly higher than that in the blank control group(t=11.240,12.330;P<0.001,0.001).Western blot results showed that the expression of VEGFR2 protein in the plasmid transfected group was significantly higher than that in the blank control group(t=8.346,P<0.01).The results of single-molecule imaging showed that the fluorescence intensity distribution of VEGFR2-GFP on the surface of RF/6A cell membrane without ligand stimulation was bimodal,in which monomer and dimer were 86.0%and 14.0%respectively.By counting the steps of GFP fluorescence bleaching,the proportions of receptor monomer,dimer,trimer,and tetramer were 81.4%,12.9%,5.5%,and 0.3%respectively.Conclusion In the absence of ligands,VEGFR2 coexists in the form of monomers and dimers on the surface of RF/6A cell membrane,and monomers are dominant.