反义RNA抑制人视网膜色素上皮细胞VEGF分泌的研究

Antisense VEGF_(121) inhibits the secretion of vascular endothelial growth factor in human RPE cell

目的 探索应用血管内皮生长因子 (VEGF12 1)的反义RNA抑制人视网膜色素上皮细胞 (RPE)VEGF表达的可行性。方法 构建编码反义hVEGF12 1真核表达质粒 ,转入人RPE细胞。将稳定表达外源基因的细胞分别置于大气氧环境 ( 2 1%O2 )或缺氧环境 ( 1%O2 )中培养 ,以空白细胞为对照 ,18h后收集上清液 ,ELISA测定其中VEGF的浓度 ,将此上清液加入人脐静脉血管内皮细胞 (HUVEC) ,观察其对细胞生长的影响。结果 反义VEGF转染能使RPE细胞VEGF的分泌量降低 62 7%。缺氧条件下 ,RPE细胞大量分泌VEGF ,18h时细胞培养上清液能刺激HUVEC细胞增殖 18 4% (P <0 0 1)。同样状态下 ,转染反义VEGF的RPE细胞培养上清液则无明显促内皮细胞生长作用 (P >0 0 1)。结论 本实验构建的反义VEGF12 1质粒能通过下调RPE细胞VEGF的蛋白分泌水平 。

ObjectiveTo identified the effect of the recombined plastid containing VEGF 121 antisense RNA on the secretion of VEGF in human retinal pigment epithelial (RPE) cells.MethodsThe plasmid (pCR3 1/anti VEGF 121 ) encoding antisense hVEGF 121 RPE cells were transfected and cultured under 21% O 2 or 1% O 2 condition. Untreated cells served as control.The concentration of VEGF in the culture medium was measured by sandwich enzyme linked immunosorbent assay (ELISA).The collected medium was also added to the human umbilical vein endothelial cells (HUVEC) to observe its effect on the growth of these cells.ResultsAntisense VEGF decreased the level of VEGF protein in the transfected RPE cells.The inhibitory rate was about 62 7%.Under hypoxia condition (1%O 2 ),medium conditioned by RPE cells contained high level of VEGF and produced a 18 4% increase in HUVEC growth at 18h( P <0 01).However, the protein level in the antisense VEGF transfected cells did not increase to the level of parental cells.The cells medium did not stimulated the growth of endothelial cells under hypoxia condition.ConclusionsThe recombinant encoding antisense VEGF 121 could successfully inhibit the expression of VEGF in RPE cells.It also suppressed the stimulatory effect of hypoxia RPE conditioned medium on the growth of HUVEC cells through the down regulation of the VEGF protein secretory rate in these cells.