腺相关病毒2-ND4基因转染细胞线粒体的研究  

Study on transfection of adeno associated virus 2-ND4 gene into mitochondria

作　　者：杨硕[1] 刘磊[1] 裴晗[1] 万幸[1] 陆朵朵 胡维琨[1] 李斌[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院眼屈光治疗中心,武汉430030

出　　处：《中华实验眼科杂志》2014年第8期693-695,共3页Chinese Journal Of Experimental Ophthalmology

基　　金：国家自然科学基金项目（30872823、8127lOl5）

摘　　要：背景Leber遗传性视神经病变（LHON）是导致视神经退行性变的线粒体遗传性疾病，主要与线粒体11778位点突变导致ND4蛋白合成异常有关，构建含正常ND4蛋白的载体是基因治疗的关键。由于ND4DNA存在于线粒体，而转染的外源基因只能进入细胞核，不能作用于突变的线粒体DNA。探讨将ND4基因成功转染到线粒体是LHON的基因治疗的关键。目的验证人工合成的ND4基因所构建的重组腺相关病毒（AAV）转染细胞后产生的ND4蛋白能否进入线粒体。方法常规体外培养转染腺病毒E1A基因的人肾上皮细胞系（293细胞），将细胞分为AAV—ND4转染组和单纯AAV转染组，分别将两种转染液加至各组的培养基中，分别于转染后12、24、36和48h用Y03量子点免疫荧光法检测细胞质中ND4的表达，细胞质中绿色荧光为ND4表达阳性。结果培养的293细胞生长良好，达到80％融合。荧光显微镜下显示，AAV-ND4基因转染组293细胞质中可见大量绿色荧光，而单纯AAV转染组仅可见线粒体蛋白红色荧光。结论AAV-ND4基因转染细胞后产生的ND4蛋白能够进入线粒体，为LHON的基因治疗提供了实验依据。Background Leber hereditary optic neuropathy （LHON） is mitochondrial DNA （mtDNA） disease and mainly leads to optical nerve degeneration. Its primary mechanism is synthesis disorder of DN4 protein due to variation of mtDNA 11778 locus. So to construct a vector with exogenous normal ND4 and transfect into mitochondria is a key of gene therapy for LHON. Objective This study was to investigate the in vitro transfection of adeno-associated virus （AAV）-ND4 gene into mitochondria. Methods Human renal epithelial cell lines transfected adenovirus E1A （293 cells） were regularly cultured and divided into two groups. Framework plasmids of recombinant AAV-ND4 or simple AAV2 were added to the cell medium respectively. The expression of ND4 in cells were located 12,24,36 and 48 hours after transfected by Y03 dual fluorescent quantum dots staining. The positive response for ND4 showed the green fluorescence. Results Cultured 293 cells grew well with 80% confluence. Abundant green fluorescence particles were seen in cytoplasm in the AAV-ND4 transfected group, but only red fluorescence from mitochondrial protein was seen in the simple AAV transfected group under the fluorescence microscope. Conclusions Exogenous ND4 protein can been successfully transfected into mitochondria using the ND4 gene constructed AAV. This result provides experimental evidence for the further study on gene therapy of LHON.

关 键 词：LEBER遗传性视神经病变 治疗 基因疗法 线粒体DNA 遗传 转染 腺相关病毒 视神经节细胞 细胞系 Y03双荧光量子点 

分 类 号：R774.6[医药卫生—眼科]