脂质体介导色素上皮衍生因子-绿色荧光蛋白质粒转染大鼠视网膜细胞及其转染途径的实验研究  

Expression of liposome-mediated PEDF-GFP gene in the retina of the BN rats through different gene delivery route

作　　者：张新秀[1] 何守志[1] 郭维维[2] 翟所强[2] 

机构地区：[1]解放军总医院眼科,北京100853 [2]解放军总医院耳鼻咽喉研究所

出　　处：《中华眼科杂志》2007年第1期55-58,共4页Chinese Journal of Ophthalmology

基　　金：全军“十五”科研基金资助项目（02M014）

摘　　要：目的观察表达质粒色素上皮衍生因子（PEDF）-绿色荧光蛋白（GFP）以脂质体介导转染入BN大鼠视网膜组织的表达及定位，探索合适的基因治疗给药方法。方法将脂质体包裹的重组表达质粒PEDF—GFP分别经玻璃体注射和视网膜下注射的途径转染至BN大鼠眼内，于一定时间内分别用荧光显微镜观察GFP表达情况，用RT—PCR方法检测PEDF表达情况。结果（1）BN大鼠视网膜下注射脂质体包裹的重组表达质粒PEDF—GFP后1d即可见在荧光显微镜下观察到在注射点周围明显的绿色荧光蛋白分布于视网膜全层包括RPE细胞层，第2、3周荧光强度比第1周增强，荧光范围也有所扩大，可以持续表达4周。RT—PCR结果证实PEDF mRNA表达明显较对照组增强。（2）经玻璃体腔注射脂质体包裹的重组表达质粒PEDF—GFP后第1天BN大鼠的视网膜和RPE细胞亦可见绿色荧光蛋白表达，且分布更均匀，范围更广。第1、2周表达明显增强，到第4周仍有表达。RT—PCR结果也证实PEDF mRNA表达明显较对照组增强。结论阳离子脂质体可以成功介导PEDF—GFP基因转染至BN大鼠的视网膜组织、RPE细胞中，并能够持续表达。视网膜下注射和玻璃体腔注射均为有效的转染途径。本研究为视网膜，脉络膜新生血管性疾病的基因治疗奠定了基础。Objective Transducing PEDF-GFP plasmid to the retina of the BN rats with cationic liposome through different gene delivery route, then observe the expression and location of the PEDF-GFP. Methods PEDF-GFP plasmid with cationic liposome was delivered to the retina of the BN rat through subrefinal injection and intravitral injection. The expression of GFP was observed under fluorescence microscope, and the mRNA of PEDF gene was detected by RT-PCR. Results Green fluorescence was emitted from the total retina include RPE cell under fluorescent microscope after 24 h in two gene delivery route, The fluorescence intensity was stronger with time changing. Gradual fluorescence increase in the retina and RPE cells occurred and lasted 4 weeks. The expression of PEDF mRNA was also detected by RTPCR after 24 h , and maintained stable 4 weeks after infection. Conclusions Cationic liposome can mediate PEDF-GFP gene into the retina of the BN rat effectively ; subretinal injection and intravitral injection both are effective gene delivery route ; and their stable expression can maintain 4 weeks after transfection.

关 键 词：脂质体 眼蛋白质类 神经生长因子 舍平类 发光蛋白质类 转染 

分 类 号：R686[医药卫生—骨科学]