新型高分子脂质体优化VEGFsiRNA转染对视网膜新生血管抑制的实验研究

Targeting VEGF siRNA transfection by new polymeric liposomes to inhibit retinal neovascularization

目的探讨新型高分子脂质体转染VEGFsiRNA对视网膜新生血管的抑制作用，评价其转染效果。方法实验研究。采用改良的Smith法将94只C57BL／6J小鼠建立氧诱导视网膜病变模型，生后11d根据分组行小鼠玻璃体腔内注射，于不同时间点，各组中随机数字表法随机选取小鼠行免疫印迹检法检测VEGF，HE染色后统计突破视网膜内界膜的血管内皮细胞核数；异硫氰酸荧光素一右旋糖酐心脏灌注后行视网膜铺片观察视网膜血管形态的变化；另各组抽取两只小鼠做视网膜冰冻切片，DAPIL核染后荧光显微镜下观察，以反映高分子脂质体携带质粒DNA的穿膜能力。结果成功建立了氧诱导视网膜病变小鼠模型。行VEGF的免疫印迹检测，17d时各组间差异有统计学意义（F=158．207，P=0．000），组间比较发现，高分子脂质体组（0．70±0．03）与脂质体组（0．66±0．04）在早期抑制效果相当（P=0．092）；22d时，各组间差异有统计学意义（F=25．695，P=0．000），组间比较显示高分子脂质体组（0．50±0．03）仍有抑制效果，与脂质体组（0．53±0．05）相比差异有统计学意义（P〈0．05）。HE染色后行视网膜新生血管内皮细胞核计数，表明生后17d时各组间差异有统计学意义（F=252．659，P=0．000），高分子脂质体组（28．0±3．44）及脂质体组（24．50＋3．06）均能有效抑制新生血管生成，生后22d时，各组间差异有统计学意义（F=193．225，P=0．000），高分子脂质体组（11．70±3．09）效果仍能显现，与脂质体组（22．90±2．60）比较差异有统计学意义（P〈0．01）。FITC心脏灌注后显示视网膜无灌注区及新生血管范围在高分子脂质体组及脂质体组明显改善，效果相当。冰冻切片显示，注射后第1天，高分子脂质体组有部分于视网膜表面表达；注射后第6天，两组GFP表达位于RPE层附近；注射后第11天，在高分子脂质体组切片中仍能找到GFP的表达。结论高分子脂质体携带的治疗基因可以减少小鼠视网膜新生血管的生成，且具有显著的穿膜能力及缓释功能，在体内可维持较长时间的治疗效果，具有临床应用的开发前景。

Objective To formulate and evaluate polymeric liposomes（PL） nanoparticles as a novel non-viral gene delivery. To explore its applicability and feasibility as a non-viral vector for gene transportation. Method Experimental study. To construct the orongoxygen induced retinopathy（OIR）mouse （C57BL/6J） model on the basis of improved Smith＇s methods. Western blot was used to measure EGF protein expression in retinal tissue at P17 and P22. HE staining and fluoreseein-dextran angiography of retinal vascular were performed to observe the morphologic alterations of retinal neovascularization. Frozen-section was used to show the membrane translocation of PL. Results In fluorescence angiograms, irregular neovascularization and fluoresce leakage were observed in O1R model. The results of Western blot showed that VEGF protein in retinal tissues were significantly different among groups（F=158.207, P=0.000）at P17 and P22（F=25.695, P=0.000）. The protein level was lower in both PL（0.70±0.03） and Lipo group （0.66±0.04） at P17 （P=0.092）, and the lower level was presented at P22 in PL group （0.50±0.03） than in Lipo group（0.53 ±0.05）（P〈0.05）. HE staining were performed to observe the significantly improvements of retinal neovaseularization in PL（28.0±3.44） and Lipo group（24.50±3.06） at P17. Moreover, inhibitory effects maintained at P22 in PL group （11.70 ± 3.09） as HE staining showed. Fluorescein angiography of retinal vaseular showed retinal non-perfusion and neovaseularization areas were smaller in both PL and Lipo group at P17. Frozen-section examination showed the property of membrane translocation. GFP expression could be seen in vitreous cavity just at first day post-intravitreal administration in PL and Lipo group, which could reaeh their peaks in external retina nearby RPE layer at P17, remaining at P22 in PL group. Conclusion The PL performed excellent ability of membrane translocation and it was a kind of slow steady released gene vector.