摘要
目的研究Ⅱ型重组腺相关病毒（recombinant adeno-associated virus 2，rAAV2）载体介导的增强型绿色荧光蛋白基因（enhanced green fluorescent protein，ECFP）对体外培养兔晶状体上皮细胞的转染和表达情况，为rAAV携带目的基因防治后囊膜混浊提供理论依据。方法组织块培养法体外培养兔晶状体上皮细胞。rAAV2-EGFP，按感染复数（multiplicity of infection，MOI）为10^3、10^4、10^5、4×10^5、10^6转染传2代细胞，转染后第1天、第3天、第5天、第7天、第9天、第11天在倒置荧光显微镜下观察晶状体上皮细胞中EGFP表达的阳性情况，记录200个细胞中EGFP阳性表达所占的百分比。当EGFP表达稳定后用激光共聚焦显微镜照相。用倒置显微镜观察rAAV转染对细胞生长和形态的影响。结果当MOI=10^3、10^4时，转染后第72小时晶状体上皮细胞中EGFP开始表达，当MOI=10^5、4×10^5、10^6时，转染后第24小时便可见EGFP阳性表达。随着MOI值的增大及时间的延长，EGFP表达效率逐渐增高，转染后第8-第9天达到高峰并维持，此时，MOI=10^3、10^4、10^5、4×10^5、10^6的转导效率分别为16％、41％、55％、86％、94％。对照组和转染组细胞的生长和形态特征无明显改变。结论腺相关病毒载体可以携带报告基因稳定转染晶状体上皮细胞，并且转染效率高，因而腺相关病毒携带目的基因防治后囊膜混浊是可行的。
Objective To investigate the expression of an enhanced green fluorescent protein （EGFP） in lens epithelial cells trannsfected with recombinant adeno-associated virus 2 （rAAV2） carrying the EGFP gene.Methods Lens epithelial cells were cultured with a tissue method in vitro. Various multiplicity of infection （MOI）（MOI = 10^3, 10^4, 10^5,4 ×10^5, 10^6）, rAAV2- EGFP were added to the second generation of lens epithelial cells. After the cells were transfected for 1, 3, 5, 7, 9 and 11 days, an inverted fluorescent microscope and a laser confocal microscope were used to detect the expression of EGFP. The percentage of positive EGFP in a random sample of 200 cells was calculated. Inverted microscope observation was performed to assess the changes in the growth and morphology of the transfected cells. Results EGFP expression in lens epithelial cells could be observed in 24 h （MOI = 10^5,4 × 10^5, 10^6） and 72 h （MOI= 10^3, 10^4） after transfection. Expression increased with an increase in MOI and reacbed the fastigium at 8 - 9 days. At that time, the transfection efficiency was 16% （MOI= 10^3）, 41% （MOI= 10^4）, 55% （MOI= 10^5）, 86% （MOI=4×10^5）and 94% （MOI= 10^6）. Transfection did not affect the growth or morphology of the cells. Conclusion An AAV vector encoding the EGFP gene can be transfected into lens epithelial cells stably and efficiently, which offers the possibility of AAV carrying a therapeutic gene to prevent posterior capsular opacification.
出处
《眼视光学杂志》
2006年第2期88-90,共3页
Chinese Journal of Optometry & Ophthalmology
关键词
腺相关病毒
绿色荧光蛋白/药效学
晶状体
上皮细胞/药物作用
后囊膜混浊
基因治疗
adeno-associated virus
green fluorescent protein/pharmacodynamices
less, crystalline
epithelial cells/drug effects
posterior capsular opacification
gene therapy
