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目的将重组表达质粒色素上皮衍生因子-绿色荧光蛋白(pigment epitheliumderived growthfactor-greenfluorescent protein,PEDF-GFP)以脂质体介导转染BN大鼠视网膜组织,观察其表达及定位。方法以脂质体介导的方法将重组表达质粒PEDF-GFP注射到BN大鼠视网膜下,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。结果在荧光显微镜下观察GFP表达情况,第1d即可见明显的绿色荧光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方法结果类似,转染后第1d即有PEDF mRNA表达,第1、2周表达明显增强,到第4周治疗眼的视网膜组织中PEDF mR-NA仍有较高水平的表达。结论阳离子脂质体可有效将PEDF-GFP基因转染视网膜组织、RPE细胞并得到持续表达,可望用于视网膜、脉络膜疾病的基因治疗。
OBJECTIVE: To investigate the expression of PEDF-GFP and its transfection into the retina of BN rats by liposome, and to observe the expression and localization of PEDF-GFP. Methods The recombinant plasmid pEDF-GFP was injected into the retina of BN rats by liposome-mediated method. The expression of GFP was observed by fluorescence microscope in a certain period of time. The expression of PEDF was detected by RT-PCR. Results The expression of GFP was observed under a fluorescence microscope. The obvious green fluorescence was observed on the entire retinal layer including the RPE cell layer on the first day. The fluorescence intensities of the second and third weeks were stronger than that of the first week, and the fluorescence range was also enlarged. 4 weeks. The result of RT-PCR was similar. The expression of PEDF mRNA was detected on the first day after transfection. The expression of PEDF mR-NA in the retinal tissue was still high at the 4th week. Conclusions Cationic liposomes can effectively transfect PEDF-GFP gene into retinal tissues and RPE cells, and can be used for the gene therapy of retinal and choroidal diseases.