摘要
目的研究2型重组腺相关病毒(recombinant adeno-associated virus2,rAAV2)载体介导增强型绿色荧光蛋白基因(enhanced green fluorescent protein,EGFP)对体外培养兔晶状体上皮细胞(N/N1003A)的转染和病毒载体对晶状体上皮细胞增生的影响,探讨rAAV2作为后囊膜混浊基因治疗载体的可行性。方法rAAV2-EGFP按感染复数(mul-tiplicity of infection,MOI)为104、105、2×105、106转染N/N1003A细胞,倒置荧光显微镜下观察晶状体上皮细胞中EGFP的表达,流式细胞仪检测rAAV2-EGFP对N/N1003A细胞的转染效率,MTT比色法测定rAAV2载体对N/N1003A增生的影响。结果EGFP在细胞中的表达呈绿色,弥漫于整个胞浆。rAAV2-EGFP的转染效率随着MOI的增加和时间的延长而增加,MOI=106时,转染后第7d达到71.12%.MTT法显示各转染组与未转染组A值无统计学差异。结论重组腺相关病毒载体可以介导EGFP基因高效稳定转染晶状体上皮细胞,并且对细胞增生无抑制作用,腺相关病毒作为后囊膜混浊基因治疗载体是可行的。
Objective To investigate the transfection efficiency of recombi- nant adeno-associated virus 2 （rAAV2）-mediated enhanced green fluorescent protein（EGFP） gene and its effects on the proliferation of rabbit lens epithelial cells（N/N1003A） in vitro, and to determine whether it is feasible to use rAAV2 as a vector in the gene therapy of posterior capsule opacification（PCO）. Methods According to various multiplicity of infection（MOI）（MOI = 10^4, 10^5 ,2 × 10^5, 10^6） ,N/N1003A cells were transfected with rAAV2 carrying EGFP gene. The expressions of EGFP was detected by inverted fluorescence microscope and the transfecion efficiency was evaluated by flow cytometry. The effect of rAAV2 vector on N/N1003A cells＇ growth was determined by HTT colorimetric assay. Results The expression of EGFP showed green fluorescence in the cytoplasm. The transfecion efficiency of the rAAV2 was increased with the increasing of HOI and the prolongation of transfection time,and reached 71.12% at 7 days（HOI = 106）. MTT showed that the A records among transfected group （with various HOI） and untransfected group had no statistical difference. Conclusion rAAV vector encoding the EGFP gene can be transfected into lens epithelial cells stably and efficiently, and the rAAV vector shows no inhibitory effect on the proliferation of N/N1003A cells in vitro, which provides the possibilitiy of rAAV as a vector in the gene therapy of PCO.
出处
《眼科新进展》
CAS
2006年第12期889-892,共4页
Recent Advances in Ophthalmology
基金
湖南省自然科学基金资助(编号:06C0040)~~
关键词
腺相关病毒
增强型绿色荧光蛋白
晶状体上皮细胞
adeno-associated virus
enhanced green fluorescent protein
lens epithelial cells
