血管内皮生长因子受体KDR基因胞外段1—3区基因转染抑制氧诱导小鼠视网膜新生血管的研究

Experimental study on inhibition of retinal neovascularisation by gene transfer of extracellular 1-3 domain of VEGF receptor KDR

目的评价脂质体介导的血管内皮生长因子受体KDR基因胞外段1-3区（KDRn3）基因转染抑制氧诱导的视网膜新生血管的效果。方法选1周龄C57Bl／6N小鼠置于氧浓度为75％±2％的氧箱中5d。回到正常环境中诱导视网膜新生血管模型。在小鼠离开氧箱的当日，向转染组小鼠玻璃体腔注射脂质体pEGFP—N1／KDRn3复合物1μl；脂质体对照组注射等量脂质体；空白对照组小鼠注射等量PBS。回到正常环境中后5d，采用视网膜铺片及视网膜冰冻切片在激光共聚焦显微镜下观察绿色荧光蛋白在视网膜的表达；采用荧光标记的右旋糖酐血管灌注下视网膜铺片方法观察视网膜新生血管的分布并测量无灌注区面积；组织学切片观察比较突破视网膜内界膜的血管内皮细胞数量。多组比较采用方差分析，有统计意义后进行两两比较的叮检验。结果转染组视网膜铺片见视网膜局部散在点状绿色荧光信号，空白对照组与脂质体对照组未见绿色荧光信号；视网膜冰冻切片见转染组视网膜神经节细胞层、内核层部分细胞胞质内见绿色荧光表达，空白对照组与脂质体对照组视网膜各层未见绿色荧光表达；视网膜铺片观察可见空白对照组与脂质体对照组在无灌注区边缘均可见新生血管芽及荧光渗漏，转染组见新生血管芽明显减少，生后第17天A、B、C3组新生血管模型小鼠各组视网膜无灌注区面积分别为（1．33±0．49）、（2．75±0．70）、（2．12±0．35）mm^2，组间比较差异有统计学意义（F=17．61，P〈0．01）。正常对照组及A、B、C组视网膜表面突破内界膜的血管内皮细胞核计数分别为0．20±0．51、13．58±2．48、23．05±3．40及21．70±2．89，组间比较差异有统计学意义（F=1085．25，P〈0．05）。结论pEGFP—N1／KDRn3基因转染可不同程度抑制氧诱导C37B1／6J小鼠视网膜新生血管的生长。

Objective To evaluate the effect of liposome mediated p lasmids KDRn3 injected into the vitreous to inhibit experimental retinal neovascularization. Methods One-week-old C57BL/6N mice were exposed to 75% ± 2% oxygen for 5 days, then returned to the room air to induce retinal neovascularization. Cationic liposome mediated KDRn3 comp-lex （ 1 μl） was injected into the vitreous in the treatment group. PBS 1 μl or liposome were injected in the control group. The pEGFP-N1/ KDRn3 expression was observed by using fluorescence microscope. Retinal neovascularization was evaluated by counting the number of vascular endothelial cell nuclei on the vitreal side of the inner limiting membrane of the retina and measuring the areas of non-perfusionsin in central retina. Results KDRn3 protein was expressed both in the ganglion layer and in the inner layer. Retinal wholemount preparation of retinal neovascular animal model showed that prominent neovascular tuft and fluorescein leakage and large areas of non-perfusionsin in central retina. Fewer neovascular tufts and fewer areas of non-perfusionsin could be seen after pEGFP-N1/KDRn3 injection. There were statistic differences between control group and pEGFP-N1/ KDRn3 injecting group with the number of vascular endothelial cell nuclei on the vitreal side of the inner limiting membrane of the retina （0. 20 ± 0. 51, 13.58 ± 2. 48,23.05 ± 3.40,21.70± 2. 89 ; F = 1085.25, P 〈 0. 05 ） and the areas of non-perfusionsin in central retina [ （ 1.33 ± 0.49 ）, （ 2. 75 ± 0. 70 ）, （ 2. 12 ± 0. 35） mm^2 ; F = 17. 61, P 〈 0. 01 ] . Conclusion pEGFP-N1/KDRn3 gene transfer can inhibit retinal neovascularisation in C57B1/6J mice of ischaemia-induced retinal neovascularisation on some extent.