血管内皮生长因子小分子干扰RNA体外实验对缺氧视网膜微血管内皮细胞的影响  

Effect of vascular endothelial growth factor small interfering RNA （siRNA） on retinal microvascular endothelial cells under hypoxia condition in vitro

作　　者：张小庄[1] 江静波[1] 罗先琼[1] 黄水清[1] 杨杰[1] 李建桥[2] 林秋雄[3] 

机构地区：[1]广东省妇幼保健院儿科,广州510010 [2]中山大学中山眼科中心 [3]广东省人民医院医学研究中心

出　　处：《中华儿科杂志》2009年第6期457-461,共5页Chinese Journal of Pediatrics

基　　金：广东省自然科学基金资助项目（032432）

摘　　要：目的探讨血管内皮生长因子小分子干扰RNA（VEGFsiRNA）体外实验对缺氧视网膜微血管内皮细胞（RMECs）的影响。方法原代培养未足月胎儿的视网膜微血管内皮细胞，用氯化钴模拟缺氧生长状态。设计、合成VEGFsiRNA并转染缺氧培养的细胞，分为VEGFsiRNA转染组（实验组）、缺氧组（空白对照组）和阴性siRNA转染组（阴性对照组），用RT-PCR和Western blot分别从mRNA和蛋白水平检测转染前后VEGF表达的变化，噻唑蓝比色法（MTY法）观察细胞的生长增殖情况。结果转染VEGFsiRNA-脂质体复合物后24h、48h、72h，hRMECsVEGFmRNA表达水平较对照组分别下降21．05％、79．67％和90．48％，hRMECsVEGF蛋白的表达水平较对照组分别下降14．58％、66．97％和81．61％。转染后12h、24h、48h及72hhRMECs的增殖分别被抑制达15．0％、42．9％、78．3％和65．9％。结论VEGFsiRNA能下调未足月胎儿缺氧视网膜微血管内皮细胞VEGF的表达，抑制视网膜微血管内皮细胞增殖，是基因治疗早产儿视网膜病变的一个研究方向。Objective To explore VEGF siRNA＇s effect on the immature fetal retinal microvascular endothelial cells in vitro. Method The fresh retinal micrangium was primarily cultured to obtain microvascular endothelial cells. CoCl2 was used to simulate oxygen-deficient conditions, siRNA directed against human VEGF was designed and chemically synthesized. There were 3 groups in our experiment： VEGF siRNA group, hypoxia control group, and negative siRNA control group. The fetal retinal micrangium vascular endothelial ceils were transfected by using liposome. The expression levels of VEGF mRNA and protein were evaluated by RT-PCR and Western blotting 24, 48, 72 h after transfection, cell proliferation was evaluated by MTF method. Result The expression levels of VEGF mRNA decreased by 21.05%, 79.67% ,and 90.48% 24 h, 48 h, and 72 h after transfection as compared to those in hypoxia control group, the expression level of VEGF protein had decreased by 14. 58% ,66. 97 %, and 81.61% as compared to those in hypoxia control group. The siRNA could decrease cell proliferation under hypoxia too, the multiplication rate after 12, 24, 48, and 72 h decreased by 15.0%, 42. 9%, 78.3% and 65.9%. Conclusion VEGF siRNA could down-regulate the expression of VEGF in immature fetal retinal microvascular endothelial cells and suppressed cell proliferation. Application of siRNA to inhibit expression of VEGF may be a hopeful way to prevent and cure ROP.

关 键 词：受体 血管内皮生长因子 RNA 小分子干扰 视网膜病 早产儿 视网膜 毛细血管通透性 内皮细胞 

分 类 号：R686[医药卫生—骨科学]