脂质体介导增强型绿色荧光蛋白基因在鼠视网膜Müller细胞中的表达  

Study on liposome-mediated enhanced green fluorescent protein expression in murine Müller cells

作　　者：熊思齐[1] 夏晓波[1] 

机构地区：[1]中南大学湘雅医院眼科,长沙410008

出　　处：《眼科研究》2009年第3期197-200,共4页Chinese Ophthalmic Research

基　　金：教育部新世纪优秀人才支持计划资助(NCET-05-0684)

摘　　要：目的研究脂质体介导增强型绿色荧光蛋白(EGFP)基因对体外培养的鼠视网膜Müller细胞转染的有效性及安全性。方法采用视网膜组织块法培养鼠视网膜Müller细胞,振荡法分离纯化细胞,并对其进行免疫细胞化学鉴定。用阳离子脂质体Lipofectamine 2000负载pEGFP-N1质粒转染Müller细胞,荧光显微镜检测转染后12、24、48、72h的转染效率。台盼蓝排斥实验检测转染和未转染细胞的活力,明确脂质体Lipofectamine 2000对Müller细胞的毒性作用。结果成功培养视网膜Müller细胞,传代后90%以上的细胞呈神经胶质纤维酸性蛋白(GFAP)染色阳性。Lipofectamine 2000介导质粒pEGFP-N1转染Müller细胞后12、24、48、72h的转染效率分别为(9.7±1.9)%、(18.1±16)%、(17.2±2.5)%、(16.9±1.7)%。台盼蓝排斥实验显示Lipofectamine 2000转染前后活细胞率分别为(98.2±5.6)%、(96.1±6.2)%。结论脂质体Lipofectamine 2000可安全有效地介导Müller细胞的转染,为进一步明确Müller细胞的病理、生理功能及靶向Müller细胞的基因治疗提供了新的手段。Objective The physiology and pathology action of retinal Mtiller cells in retinal metabolism is much concerned. Recent year, Mtiller cells become a target topic in the study on ophthalmology and treatment of ocular disease. Present study was to evaluate the efficacy and security of liposome transferring enhanced green fluorescent protein（EGFP） to cultured retinal Mvller cells of mouse. Methods Retinal MUller cells of mouse were isolated and cultured by tissue inoculation in complete medium. The second passage of cells were identified by glial fibrillary acidic protein （ GFAP）. The mixture of 50 uL DMEM ＋ 2 uL Lipofectamine TM2000 and 50uL DMEM ＋ 1.0 ug pEGFP-NI was added into cultured cells for the transfection of cells and the transfection efficiency of Lipofectamine 2000 was detected 12,24,48 and 72 hours after transfection. The viability of transfected and untransfected cells was measured by trypan blue rejection test. Results 90% cultured retinal Mailer ceils were positively reacted for GFAP. Lipofectamine 2000 ＋ EGFP-transfected ceils presented the green influorenscence with the transfeetion efficiency （9.7 ± 1.9）%, （18.1 ± 16）%, （17.2 ± 2.5）%, （16.9 ± 1.7）% at 12, 24,48 and 72 hours respectively, showing a statistically significant difference among the groups （ P 〈 0. 01 ）. The viability of transfected and untransfected cells was （98.2 ± 5.6）% , （96. 1 ± 6.2）% respectively with a significant difference between them（ P 〈 0.01 ）. Conclusion Lipofectamine 2000 can transfer exogenous gene to retinal MUller cells effectively and safely,which may provide a novel tool in the invastigation of physiological and pathological function of Mailer cells and treatment of some ocular diseases.

关 键 词：MÜLLER细胞 脂质体 转染 增强型绿色荧光蛋白基因 

分 类 号：R774[医药卫生—眼科]