中文摘要：

背景内皮抑素（ES）是目前已知最强的内源性血管形成抑制因子，能抑制新生血管的发生，而精氨酸-甘氨酸-天冬氨酸（RGD）序列的聚合能增强Es基因的功能。目的探讨改良Es基因对大鼠角膜新生血管（CNV）中血管内皮生长因子（VEGF）及VEGF受体2（Flk-1）表达的影响及作用机制。方法102只清洁级sD大鼠按随机数字表法分为模型组、pCI空载体组、pCI—ES转染组、pCI—RGDRGD—ES转染组，每组24只；正常组6只。用浸有1mol／LNaOH溶液的直径3mm圆形滤纸片快速贴于大鼠角膜中央40s建立角膜碱烧伤CNV模型，以pCI质粒作为改良前后E5基因的表达载体，每周2次结膜下注射3μg含质粒的转染液进行基因治疗。每日裂隙灯显微镜下观察角膜水肿及CNV生长情况，计算CNV面积。于碱烧伤后1、4、7、14d用过量麻醉法处死模型鼠并获取角膜组织，用免疫组织化学法检测CNV内皮细胞中CD34的表达以计算CNV密度，通过逆转录聚合酶链反应（RT—PCR）及Westernblot法分别检测角膜VEGFmRNA及Flk-1在角膜中的表达。结果角膜碱烧伤后7～14d，pCI—ES转染组、pCI—RGDRGD—ES转染组CNV面积均明显小于pCI空载体组和模型组，CNV密度均明显小于pCI空载体组和模型组，差异均有统计学意义（P〈0．05，P〈0．01）；pCI—RGDRGD—ES转染组CNV面积均明显小于pCI—ES转染组，CNV密度均明显少于pCI—ES转染组，差异均有统计学意义（P〈0．05，P〈0．01）。碱烧伤后4d，pCI—ES转染组、pCI—RGDRGD—ES转染组大鼠角膜Flk-1蛋白表达水平明显低于正常对照组大鼠，差异有统计学意义（P〈O．05）；碱烧伤后4d，pCI—ES转染组、pCI—RGDRGD—Es转染组大鼠角膜VEGFmRNA的表达水平明显低于正常对照组大鼠，差异有统计学意义（P〈O．05）；pCI—RGDRGD—ES转染组大鼠角膜VEGFmRNA表达水平明显低于pCI—ES转染组大鼠，差异有统计学意义（

英文摘要：

Background Endostatin （ES） is currently the strongest endogenous angiognesis inhibitor,and it can inhibit the occurrence of neovascularization. Various studies demonstrated that the poly RGD sequence can enhance the function of the ES gene. Objective This study was to evaluate the use of gene therapy of modified ES for alkaline burn-induced corneal neovascularization （ CNV）. Methods One hundred and two clean SD rats were randomly divided into the normal control group, the pCI empty vector group, the pCI-ES group, and the pCI-RGDRGD- ES group. Corneal neovascularization models were established by placing a piece of 3 mm filter paper with 1 mol/L N aOH at the central cornea for 40 seconds. 3 μg of the pCI blank vector, ES-tranfected pCI blank vector, or RGDRGD- ES-transfected pCI vector was injected into the superior bulbar conjunctiva after the alkali burn twice at 1-week intervals. Area of CNV and edema of the cornea in the various groups of rats were examined daily under the slit lamp biomicroscope. 1,4,7 and 14 days after operation, the rats were sacrificed by the excessive anesthesia method and corneal tissues were obtained to evaluate pathological changes. The expression of CD34 in vascular endothelial cells was detected by immunochemistry to calculate the corneal neovascular density. The expressions of VEGF mRNA and Flk-1 protein in the corneas were detected by reverse transcription polymerase chain reaction （RT-PCR） and Western blot analysis. The use and maintenance of animals followed the Statement of ARVO. Results Seven to fourteen days after corneal alkali-burning,the corneal neovascular area was smaller in the pCI-ES group and pCI-RGDRGD-ES group compared with the normal control group and pCI blank vector group （ P〈0. 05 ,P〈0. 01 ） ,and nevascular area inthe pCI-RGDRGD-ES group was smaller than that in the pCI-ES group （P〈0. 05）. The expression level of CD34 was significantly lower in the pCI-ES group and pCI-RGDRGD-ES group than that in the normal control group and pCI blank