CAJ | 학술논문

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ND4 DNA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48 h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.
배경 Leber유전성시신경병변(LHON)시도치시신경퇴행성변적선립체유전성질병,주요여선립체11778위점돌변도치ND4단백합성이상유관,구건함정상ND4단백적재체시기인치료적관건.유우ND4 DNA존재우선립체,이전염적외원기인지능진입세포핵,불능작용우돌변적선립체DNA.탐토장ND4기인성공전염도선립체시LHON적기인치료적관건. 목적 험증인공합성적ND4기인소구건적중조선상관병독(AAV)전염세포후산생적ND4단백능부진입선립체.방법 상규체외배양전염선병독E1A기인적인신상피세포계(293세포),장세포분위AAV-ND4전염조화단순AAV전염조,분별장량충전염액가지각조적배양기중,분별우전염후12、24、36화48 h용Y03양자점면역형광법검측세포질중ND4적표체,세포질중록색형광위ND4표체양성. 결과 배양적293세포생장량호,체도80％융합.형광현미경하현시,AAV-ND4기인전염조293세포질중가견대량록색형광,이단순AAV전염조부가견선립체단백홍색형광.결론 AAV-ND4기인전염세포후산생적ND4단백능구진입선립체,위LHON적기인치료제공료실험의거.
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.