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

发布时间：2018-11-03 21:27

【摘要】：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(P0.05),且MOI增加时EGFP表达增强(P0.05)。E组荧光强度明显高于A、B、D组(P0.05),但与C组比较差异无统计学意义(P0.05)。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。
[Abstract]:Objective to observe the transfection efficiency and toxicity of enhanced green fluorescent protein (LV-EGFP) mediated by lentivirus vector in rat cornea at different concentrations and pathways, and to explore the best way to transfect cornea by lentivirus-mediated green fluorescent protein (LV-EGFP). Methods Twenty-five SD rats were randomly divided into five groups: group A was infected with complex (MOI) = 5 point eye group, group B was injected under MOI=5 conjunctiva group, group C was MOI=10 point eye group, group D was subconjunctival injection group, group E was MOI=10 transfection group in vitro. The corneal fluorescence distribution was observed under fluorescence microscope and the fluorescence intensity was analyzed after transfection with corresponding concentration of LV-EGFP.7d. The pathological changes of corneal cells after transfection were observed by HE staining. Results fluorescence microscope showed that the distribution of corneal fluorescence in the same MOI group was more uniform than that in the subconjunctival injection. The values of relative fluorescence intensity in each group were: group A, 0.1803 卤0.0440, group B, 0.1061 卤0.0434A, group C, 0.2369 卤0.0157, group D, 0.2002 卤0.0307, group E, 0.2434 卤0.0173; The fluorescence intensity of cornea of point eye mode was higher than that of subconjunctival injection (P0.05), and the expression of EGFP was increased with the increase of MOI (P0.05). The fluorescence intensity of). E group was significantly higher than that of Aconjunctival injection group (P0.05). However, there was no significant difference between group C and group C (P0.05). HE staining showed that the corneal cells in each group were normal, no obvious apoptotic cells were found. After 7 days of transfection in vitro, corneal endothelial cells grew well, without shrinkage, deformation and deletion. Conclusion LV-EGFP can effectively transfect the cornea of rats at low MOI and the safety of continuous transfection is good. The transfection efficiency of point-eye method is higher than that of subconjunctival injection, and MOI can improve the efficiency of corneal transfection.
【作者单位】：重庆医科大学附属第一医院眼科重庆市眼科学重点实验室;
【基金】：国家自然科学基金(81170822)~~
【分类号】：R772.2

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