抗血管内皮生长因子小干扰RNA抑制大鼠角膜新生血管形成的研究

目的 探讨抗血管内皮生长因子小干扰RNA(VEGF-siRNA)对角膜碱烧伤急性期新生血管(CNV)形成的抑制作用.方法 实验研究.体外化学合成VEGF序列特异性双链小干扰RNA(VEGF-siRNA),脂质体介导转染体外培养的大鼠角膜上皮和基质细胞,实时PCR法检测转染后各时段VEGF mRNA表达水平,酶联免疫吸附实验测定转染后各时段VEGF蛋白表达水平.制作大鼠角膜碱烧伤动物模型,采用VEGF-siRNA转染的角膜上皮移植联合前房注射脂质体包裹的VEGF-siRNA治疗急性期CNV,免疫组化和ELISA分别检测角膜VEGF表达,观察术后CNV形成.细胞实验和动物实验中siRNA处理组和对照组VEGF蛋白含量比较,采用两样本均数比较的t检验.结果 siRNA对角膜上皮细胞VEGFmRNA的抑制效率达60%～84%,角膜基质细胞VEGFmRNA的抑制效率达59%～76%,角膜上皮和基质细胞VEGF蛋白表达也有相应的下降.采用VEGF-siRNA转染的角膜上皮移植联合前房注射VEGF-siRNA治疗,实验组CNV面积显著小于对照组;实验组VEGF表达水平显著低于对照组.结论 VEGF-siRNA能显著抑制大鼠角膜碱烧伤急性期CNV形成.

Small interference RNA targeting vascular endothelial growth factor inhibits rat corneal neovascularization

Objective To test the effects of VEGF-siRNA-transacted corneal epithelium on corneal neovascularization(CNV).Methods It WaS all experimental study.Cultured rat corneal epithelial cells and keratocytes were transfected with synthesize VEGF siRNA by lipofeetamine 2000.The level of VEGF mRNA was analyzed by real time PCR,and the protein levels were determined by enzyme-linked immunosorbnent assay(ELISA).CNV WaS induced by cauterization with 1 mol/L sodium hydroxide in rat corneas.The VEGF-siRNA-transfected-comeal epithelium cells were transplanted to the CNV lesions.Immediately after transplantation,the VEGF-siRNA combined with lipofectamine 2000 were directly transfected rat cornea through injecting into the anterior chamber.After surgery,the surface areas occupied by new vessels were measured,and VEGF protein was localized by immunohistochemistry.Results The levels of VEGF expression at both mRNA and protein in the VEGF-siRNA transfected corneal epithelial cells and keratocyws were significantly Jower than those of control cells.VEGF siRNA could inhibit the expression of VEGF mRNA in C01Tleal epithelial cells and keratocytes to 57%-85%and 59%-78%,respectively.The VEGF-siRNA-transfected-corneal epithelium transplantation significantly decreased the surface areas occupied by new vessels.VEGF expression level in interference groups was lower than that in the control group.Conclusions The development of CNV is markedly suppressed by VEGF-siRNA transfection in vivo.