慢病毒载体介导增强型绿色荧光蛋白转染大鼠角膜的效率及其毒性研究

Effectiveness and toxicity of the transfection of lentiviral vector-mediated enhanced green fluorescent protein to rat cornea

目的观察慢病毒载体介导的增强型绿色荧光蛋白(LV-EGFP)经不同浓度和途径转染大鼠角膜的转染效率及毒性,探讨慢病毒转染角膜的最佳途径。方法 25只SD大鼠,随机分为5组。A组为感染复数(MOI)=5点眼组,B组为MOI=5结膜下注射组,C组为MOI=10点眼组,D组为MOI=10结膜下注射组,E组为MOI=10离体转染组,各组予以相应浓度LV-EGFP。7d后取转染后角膜于荧光显微镜下观察荧光分布,并分析荧光强度;HE染色观察转染后角膜细胞病理改变。结果荧光显微镜观察显示,相同MOI组之间比较,点眼方式的角膜荧光分布较结膜下注射方式均匀。各组相对荧光强度值分别为:A组0.1803±0.0440,B组0.1061±0.0434,C组0.2369±0.0157,D组0.2002±0.0307,E组0.2434±0.0173,点眼方式的角膜荧光强度高于结膜下注射方式(P<0.05),且MOI增加时EGFP表达增强(P<0.05)。E组荧光强度明显高于A、B、D组(P<0.05),但与C组比较差异无统计学意义(P>0.05)。HE染色显示各组角膜细胞形态正常,未见明显凋亡细胞。离体转染角膜经过7d培养液转染后,角膜内皮细胞生长良好,未出现皱缩、变形及缺失等病理改变。结论 LV-EGFP可在较低MOI下有效转染大鼠角膜,且持续转染的安全性良好;点眼方式的转染效率高于比结膜下注射,提高MOI能提高角膜转染的效率。

Objective To investigate the effectiveness and toxicity of the transfection of lentiviral vector-mediated enhanced green fluorescent protein （LV-EGFP） to rat cornea with different concentrations and methods, and to look for the most effective way for transfecting the virus to rat cornea. Methods Twenty-five SD rats were randomly divided into five groups, namely MOI=5 eyedrop group （group A）, MOI=5 subconjunctival injection group （group B）, MOI=10 eyedrop group （group C）, MOI=10 subconjunctival injection group （group D）, and MOI=10 in vitro transfection group （group E）. Rats in each group were treated with corresponding concentration of LV-EGFP respectively. The distribution and intensity of fluorescence in the corneas were observed under inverted fluorescence microscope, and the corneal cellular morphology was observed with HE staining 7 days after transfection. Results Within the same MOI groups, fluorescence spread more uniformly in eyedrop groups as compared with that in subconjunctival injection groups. The fluorescence intensity （A value） in five groups were 0.1803± 0.0440, 0.1061 ± 0.0434, 0.2369 ± 0.0157, 0.2002 ± 0.0307, 0.2434 ± 0.0173 respectively, which was stronger in eyedrop groups than in subconjunctival injection groups （P〈0.05）. In high MOI groups more EGFP could be expressed as compared with low MOI groups （P〈0.05）. The fluorescence intensity was significantly higher in group E than in groups A, B and D, while there was no statistical significant difference between groups E and C. HE staining showed no apoptotic corneal endothelial cells. The corneal endothelial ceils were still growing well with no shrinkage, deformation and loss in vitro 7 days after transfection. Conclusion Rat cornea could be effectively transfected by LV-EGFP with low MOI. Eyedrop method is found to have a higher efficiency compared with subconjunctival injection. An increase in amount of MOI can increase transfection efficiency. Continuous transfection with low MOI to cornea of rat is safe.