抗血管生成作用的VEGFxxxb在缺氧视网膜色素上皮细胞中的表达

Expression of anti-angiogenic vascular endothelial growth factor isoforms VEGFxxxb in human retinal pigment epithelium cells

背景 缺氧可诱导视网膜色素上皮（RPE）细胞分泌血管内皮生长因子A（VEGF-A）,从而促进脉络膜新生血管（CNV）形成.VEGF-A依选择性剪接方式的不同,可形成具有促血管生成作用的VEGFxxx和具有抗血管生成作用的VEGFxxxb,但后者在常氧和缺氧状态下在人RPE细胞中的表达变化及其作用尚不清楚.目的 从mRNA水平和蛋白水平检测模拟缺氧状态培养的RPE细胞中VEGFmb的表达,研究VEGFxxxb在常氧和缺氧培养的RPE细胞中的表达变化.方法 以人RPE细胞系ARPE-19为研究对象,将150 μmol/L化学诱导剂CoC12加入细胞培养液制备缺氧细胞模型,分别于0h（常氧培养）及缺氧培养3、6、12和24 h收获细胞或细胞上清液,采用逆转录PCR（RT-PCR）法检测细胞内VEGFxxxmRNA和VEGFxxxb mRNA的表达;采用Western blot法检测细胞内总VEGF-A蛋白和VEGFxxxb蛋白的表达;采用ELISA法检测细胞上清中总VEGF-A蛋白和VEGFxxxb蛋白的含量.结果 在常氧及诱导缺氧后,ARPE-19细胞中均可检测到VEGFxxxmRNA和VEGFxxxb mRNA的表达,检测出的VEGFxxxmRNA中同时出现VEGF121、VEGF165和极微弱的VEGF189条带;检出的VEGFxxxb mRNA中可见VEGF165b条带.缺氧培养的细胞中随着缺氧时间的延长,VEGFxxxmRNA显示出表达逐渐增加的趋势,而VEGFxxxb mRNA则显示出表达逐渐减少的趋势.Western blot法可检测到细胞中VEGFxxxb蛋白的表达,尤以VEGF165b表达最强,随缺氧培养时间的延长,VEGF165b蛋白表达逐渐减少.培养的细胞上清中VEGFxxxb蛋白质量浓度由常氧培养时的（166.82±2.55） pg/ml下降至缺氧培养24 h的（125.35±2.10）pg/ml,而总VEGF-A蛋白则由（294.27±11.97） pg/ml上升至（582.26±12.98） pg/ml.细胞中VEGFxxxbb蛋白占总VEGF-A蛋白的比例随缺氧培养时间的延长,由常氧培养条件下的（56.71±1.02）％逐渐下降至缺氧培养24 h的（21.53±0.08）％.结论 ARPE-19细胞在常氧和缺氧培养条件下均可检测到VEGFxxxbb mRNA和蛋白的表达.缺氧培养后ARPE-19细胞中VEGFxxx mRNA的表达水平逐渐降低.常氧培养时细胞中总VEGF-A蛋白以VEGFxxxbb占主导,而缺氧后VEGFxxxbb占总VEGF-A比例下降.

Background Hypoxia can increase the secretion of vascular endothelial growth factor-A （VEGF A） by retinal pigment epithelium （RPE） cells and initiate choroidal neovascularization （CNV） consequently.Two VEGF-A isoforms,the angiogenic VEGF-A family （VEGFxxx） and the anti-angiogenic VEGF-A family （VEGFxxxb）,are found and formed by alternative splicing.However,the expressing changes and its effect in human RPE cells under the normoxia and hypoxia are unclear.Objective This study was to investigate the expression of VEGFxxx b in human RPE cells under hypoxia.Methods ARPE-19,a human RPE cell line,were cultured.CoCl2 150 μmol/L was added into the medium to mimic the hypoxia environment for 0 hour （normoxia group）,3,12 and 24 hours to induce the hypoxia cells.The cells or suspension was harvested at various time points.The expressions of VEGFxxx mRNA and VEGFxxxb mRNA in the cells were detected,and the expressions of the total VEGF-A protein and VEGFxxxb protein in the cells were assayed by Western blot.ELISA was used to determine the contents of total VEGF-A and VEGFxxxb proteins in suspension.Results VEGFxxx mRNA and VEGFxxxb mRNA were expressed in the ARPE-19 cells in both normoxia and hypoxia,and multiple VEGFxxx mRNA isoforms appeared,including VEGF121 mRNA,VEGF165 mRNA and extremely faint VEGF189 mRNA,but only VEGF165b band was seen in VEGFxxxb mRNA.With the prolong of hypoxia culture,the expression of VEGFxxx mRNA showed gradual increase,while VEGFxxx b mRNA appeared gradual decrease.Western blot exhibited that VEGFxxxb was expressed in the cells cultured by normoxia and hypoxia with the strongest expression in VEGF165b.The VEGFxxxb content in suspension of medium was （166.82± 2.55） pg/ml under the normoxia environment and （125.35 ±2.10） pg/ml in 24 hours under the hypoxia,and the total VEGF-A protein elevated from （294.27 ± 11.97） pg/ml under the normoxia environment to （582.26 ± 12.98） pg/ml in 24 hours under the hypoxia.In addition,the proportion of VEGFxxxb to total VEGF-A in the cells lowed from （56.71 ± 1.02） % under the normoxia environment to （21.53 ±0.08） % in 24 hours under the hypoxia.Conclusions VEGFxxxb isoforms are expressed in both normoxia and hypoxia ARPE-19 cells.VEGFxxxb isoform is a predominant isoform in normoxia ARPE-19 cells.In hypoxia ARPE-19 cells,however,the expression of VEGF A is significantly stronger than that of VEGFxxxb.