Caveolin-1对OGD/R人脐带血内皮祖细胞中VEGF表达的影响

Influence of Caveolin-1 on VEGF Expression in OGD/R Human Umbilical Cord Blood Endothelial Progenitor Cells

通过探讨caveolin-1对氧糖剥夺/复氧(oxygen glucose deprivation/reoxygenation,OGD/R)人脐带血内皮祖细胞(endothelial progenitor cells,EPCs)中血管内皮生长因子(vascular endothelial growth factor,VEGF)的影响,为caveolin-1在脑缺血缺氧性疾病中的应用提供实验依据。采用密度梯度离心法获取脐带血单个核细胞,接种在鼠尾I型胶原包被的培养板中,用内皮细胞培养液EGM-2培养,观察EPCs生长情况,通过观察细胞形态、双荧光染色法、免疫荧光细胞化学染色法及流式细胞仪等技术对培养的EPCs进行鉴定。选取EPCs高表达的时间点,构建针对caveolin-1基因的RNA干扰真核表达质粒,建立基因沉默和OGD/R模型,随机分为正常对照组、OGD/R组、caveolin-1基因沉默组、caveolin-1基因沉默OGD/R组。免疫荧光法和Western blot检测caveolin-1及VEGF的表达。人脐带血可分离获得内皮祖细胞;成功构建caveolin-1基因沉默模型;caveolin-1及VEGF的表达在caveolin-1基因沉默组显著低于正常对照组(P<0.01);OGD/R组caveolin-1及VEGF表达明显高于正常对照组(P<0.01);OGD/R组caveolin-1及VEGF表达明显高于caveolin-1基因沉默OGD/R组(P<0.01);caveolin-1基因沉默组caveolin-1和VEGF表达量高于caveolin-1基因沉默OGD/R组(P<0.05)。以上结果表明,caveolin-1能够影响OGD/R的EPCs中VEGF的表达,是促进血管细胞损伤修复的可能因素之一。

The purpose of this study was to provide experimental basis for the application of caveolin-1 in cerebral hypoxic-ischemic diseases through investigating the influence of caveolin-1 on VEGF expression in OGD/R（oxygen glucose deprivation/reoxygenation） human umbilical cord blood endothelial progenitor cells（EPCs）. Density gradient centrifugation was adopted to obtain mononuclear cells from the cord blood, which were then inoculated to the culture plates coated with rat tail collagen-I and cultured using the endothelial cell nutrient solution of EGM-2. The proliferation and growth of EPCs were observed and identified through cellular morphology observation, double fluorescence staining, immunofluorescence cytochemistry staining, flow cytom-etry, etc. The time points when EPCs were highly expressed were chosen to establish the caveolin-1-targeted RNA interference eukaryotic plasmids and build gene silencing and OGD/R models which were then randomly grouped into the control group, the OGD/R group, the caveolin-1 gene silencing group and the caveolin-1 gene silencing OGD/R group. Immunofluorescence and Western blot were adopted to detect the expression of caveolin-1 and VEGF. EPCs could be separated and obtained from human umbilical cord blood; caveolin-1 gene silencing models were successfully constructed; the expression of caveolin-1 and VEGF in the caveolin-1 gene silencing group was significantly lower than that in the control group（P〈0.01）, but higher than that in the caveolin-1 gene silencing OGD/R group（P〈0.05）; and the expression of caveolin-1 and VEGF in the OGD/R group was significantly higher than that in the control group and caveolin-1 gene silencing OGD/R group（P〈0.01）. The results showed that caveolin-1 could affect the expression of VEGF of OGD/R EPCs, which should be one of favorable factors for vascular cytothesis.