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

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)mm2,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)mm2,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)mm2,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.

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

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) mm2 in the experimental group and (1.96±0. 34) mm2 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) mm2 in the experimental group which was obviously higher than (1.26±0. 38) mm2 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)mm2 and (0.41±0.02)mm2 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.