腺病毒对人视网膜色素上皮细胞转染腺相关病毒的增强作用  

Enhance efficiency of adenovirus on AAV transfering human RPE cells

作　　者：李惠明[1] 季迅达[1] 王慧萍[1] 王丰[1] 韦芳[1] 陈霞芳[1] 王煜非[1] 黄倩[1] 

机构地区：[1]上海交通大学附属第一人民医院中心实验室,200080

出　　处：《中华实验眼科杂志》2011年第1期21-25,共5页Chinese Journal Of Experimental Ophthalmology

基　　金：国家自然科学基金“杰出青年”基金项目（30325043）、上海市自然科学基金项目（09ZH1425000）、上海市视觉复明中心基金项目（FM040102-3）

摘　　要：背景腺相关病毒（AAV）作为治疗载体能够长期稳定地表达治疗基因，但对视网膜色素上皮（RPE）细胞的转染效率较低，探讨提高AAV对RPE细胞转染效率的方法是目前研究的热点之一。目的探讨少垃腺病毒对AAV转染RPE细胞的作用。方法从成人供体眼杯巾以胰蛋白酶消化法分离RPE细胞，并将其在含有质量分数10％胎牛血清的DMEM培养液中原代培养30d。采用携带报告基因增强型绿色荧光蛋白（EGFP）的2型血清型AAV2 1×10^4感染复数（MOI）转染细胞作为AAV2-EGFP组，在加入上述物质的同时，加入不同滴度的非复制型腺病毒（Ad-null）作为AAV2-EGFP＋Ad-null组。转染后采用倒置荧光显微镜观察并计数EGFP阳性细胞，采用Westernblot法检测转染细胞巾EGFP表达量的变化。结果分离的RPE细胞富含色素颗粒。AAV2-EGFP转染RPE细胞后第2天EGFP开始表达，至第12天达高峰，之后维持在较高的水平至23d。荧光显微镜下可见随着Ad-null滴度的增加，AAV2-EGFP＋Ad-null组中各滴度时EGFP阳性的RPE细胞数和平均荧光强度显著增加，显示出明显的剂量效应关系。联合0．1MOI的Ad-null即可见显著的增强作用，至10MOI时达高峰，但当Ad-null滴度提高至100MOI以上即出现显著的细胞毒性作用。Westernblot检测显示，EGFP在细胞中的蛋白表达水平AAV2-EGFP＋Ad-null组较AAV2-EGFP组明显提高。结论联合使用少量腺病毒可明显增强AAV对人RPE细胞的转染，为今后应用AAV作为载体携带外源基因治疗视网膜色素变性和新生血管性疾病提供了实验依据。Background Adeno-associated virus-based vector is one of most efficient vehicles. It presents with a long-term and efficient transfer and expression of therapeutic genes with minimal toxicity. But its delayed-and low-efficient transgene expression limits the application of AAV vector. To explore an improving method of AAV infecting RPE cells is the hot spot. Objective Present study was to investigate whether adeno-associated virus （AAV） combined with low dose non-replicable adenovirus （Ad-null） can enhance its infection efficiency on RPE cells in vitro. Methods Human RPE cells were isolated from the donate eyeballs under the approval of the Ethic Committee of this hospital. The cells were cultured in DMEM containing 10% fetal bovine serum. AAV particles with enhanced green fluorescence protein （EGFP） were added into the medium alone or in combination with different amount of adenovirus for 30 days. The cells were detected under the fluorescence microscope, and the protein expression levels of report gene EGFP in RPE cells were analyzed with Western blotting assay. Results Melanin granules could be found in cultured RPE cells. EGFP was expressed in RPE cells at 2 days afler AAV-EGFP infection and peaked at 12 days and remained for about 3-week duration, showing the green influorescence under the influorescence microscope. After the cells were infected by AAV2-EGFP with 0.01 to 1000 MOI Ad-null respectively, the number of cells with green influorescence was obviously increased with the enhanced influorescence intensity. The enhance of the infection efficiency began in the 0. 1 MOI Ad-null group and peaked in 10 MOI Ad-null group. Dead cells were exhibited in the 100 or more MOI Ad-nulor group. Western blotting assay demonstrated that the protein expression level of EGFP in RPE cells enhanced significantly in 1 and 10 MOI Ad-null groups compared with only AAV infection group. Conclusion These finding suggested that the infection efficiency of AAV can be improved significantly when it is used with low （

关 键 词：视网膜色素上皮细胞 腺病毒 腺相关病毒 基因疗法 

分 类 号：R77[医药卫生—眼科]