重组腺相关病毒-色素上皮衍生因子抑制氧诱导小鼠视网膜新生血管的作用

Inhibitory effects of rAAV2-pigment epithelial derived factor on oxygen-induced retinal neovascularization in mice

目的 观察重组腺相关病毒-色素上皮衍生因子（rAAV2-PEDF）对氧诱导小鼠视网膜新生血管（RNV）的作用.方法 选择3日龄C57/BL6小鼠22只,左眼为实验服,右眼为对照眼,微量注射器玻璃体腔分别注射rAAV2-PEDF和rAAV2-绿色荧光蛋白1μl.注射后立即将小鼠放入氧箱,建立氧诱导血管增生性视网膜病变模型.取13日龄小鼠4只提取视网膜总蛋白,蛋白质免疫印迹法（Western blot）检测色素上皮衍生因子（PEDF）蛋白表达.17日龄小鼠12只,荧光素心脏灌注视网膜铺片观察血管形态和分布.17日龄小鼠6只,视网膜冰冻切片外源凝集素标记后染色观察血管形态和分布.Image-Pro Plus5.1软件测量分析无荧光素灌注区和RNV的绝对面积和相对面积.结果 实验眼PEDF蛋白表达显著高于对照眼.视网膜铺片定量结果显示,实验眼和对照跟绝对无灌注区面积分别为（0.96±0.22）、（1.96±0.34）mm^2,差异有统计学意义（t=-8.554,P〈0.01）;相对无灌注区面积分别为（8.64±1.52）%、（17.27±2.98）%,差异有统计学意义（t=-8.97,P〈0.01）.实验眼和对照眼绝对新生血管面积分别为（0.37±0.11）、（1.26±0.38）mm^2,差异有统计学意义（t=-7.8,P〈0.01）;相对新生血管面积分别为（3.96±0.66）%、（11.45±2.06）%,差异有统计学意义（t=-8.51,P〈0.01）.外源凝集素标记定量结果显示,实验眼和对照眼RNV面积分别为（0.11±0.003）、（0.41 4-0.02）mm^2,差异有统计学意义（t=-5.14,P〈0.01）.结论 rAAV2-PEDF可成功转染小鼠视网膜组织并稳定表达PEDF蛋白,不仅可以减少氧诱导血管增生性视网膜病变小鼠视网膜无灌注面积,而且可显著抑制RNV生成.

Objective To investigate the effects of recombinant adeno-associated virus type-2 （rAAV2） mediated delivery of pigment epithelium-derived factor （PEDF） on oxygen-induced retinal neovascularization （OIRNV） in mice. Methods A total of 22 C57/BL6 mice at the age of 3 days received intravitreal injections of 1 μl rAAV2-PEDF and rAAV2-EGFP into the left eyes （experimental group） and the right eyes （control group）. All mice were put into the oxygen box right after the injection to induce the OIRNV model. 4 mice were sacrificed and PEDF protein in retina was measured by western blot at postnatal days 13 （P13）. Twelve mice underwent retinal angiography with high molecular weight fluorescein-dextran,and another 6 mice were sacrificed for retinal lectin immunohistochemistry staining at P17. Absolute and relative non-perfusion areas of retinal neovascularization were analyzed by Image-Pro Plus 5.1 software.Results The expression level of PEDF protein was higher in the experimental group than that in the control group. The absolute non-perfusion area was （0. 96 ＋ 0.22） mm^2 in the experimental group and （1.96±0. 34） mm^2 in the control group; the difference between the two groups was significant （t = -8. 554, P〈0.01）. The relative non-perfusion area was （8. 64 ± 1.52） % in the experimental group and （17. 27 ± 2. 98）% in the control group with a significant difference between the two groups （t = -8. 97, P〈0. 01）. The absolute area of retinal neovascularization was （0. 37 ± 0. 11） mm^2 in the experimental group which was obviously higher than （1.26±0. 38） mm^2 in the control group （t=-7. 8, P〈0. 01）; the relative areas in experimental and control groups was （3. 96 ± 0. 66） % and （ 11.45 ± 2. 06） %, respectively, whose difference is apparently （t=-8. 51, P〈0. 01）. The areas of retina neovascularization were （0. 11±0. 003）mm^2 and （0.41±0.02）mm^2 in the experimental and control groups, respectively, and the differencebetween the two groups was significant （t =- 5.14, P〈 0. 01）. Conclusions PEDF protein can stably express in the mice retina after rAAV2-PEDF transfetion, rAAV2-PEDF can decrease the retinal nonperfusion areas and inhibit the retinal neovascularization in OIRNV mice.