重组腺病毒介导绿色荧光蛋白基因对小鼠眼组织的转染和表达  

Recombinant adenovirus-mediated transfection and expression of enhanced green fluorescence protein in mouse anterior chamber

作　　者：于慧[1] 吴继红[1] 张圣海[1] 刘新建[1] 张巨峰[1] 易苗英[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：《眼科研究》2007年第12期904-907,共4页Chinese Ophthalmic Research

基　　金：国家自然科学基金"杰出青年项目"(30325043);上海市卫生局医学领军人才基金项目(LJ06036);上海市科学技术委员会"登山计划项目"(064119539)资助

摘　　要：目的研究不同剂量Ad5-EGFP前房注射后,报告基因在小鼠眼部组织的表达情况及分布特点。方法24只BALB/c小鼠随机分组。实验组以不同浓度梯度前房注射Ad5-EGFP。对照组前房分别注射PBS和不给予任何注射。观察活体眼部组织EGFP表达情况及阳性细胞的类型。结果低浓度注射组在各个时间点眼表均未观察到EGFP荧光表达。高浓度注射组在注射病毒第1d即可以观察EGFP的表达,在第10d时眼表EGFP荧光亮度、范围增加最为明显。EGFP在角膜内皮细胞、虹膜色素上皮细胞及小梁网阳性表达。结论Ad5型腺病毒载体定向携带报告基因在眼前节表达,为眼前节疾病的基因治疗提供了实验依据。Objective Anterior chamber is thought to be a main target segment in gene therapy of the anterior eye diseases. As the special immune characteristics of anterior chamber, there are few data available regarding replication-defective adenovirus-mediated report gene in anterior chamber. Present study was to explore the expression and distribution of enhanced green fluorescence protein（EGFP） transduced into Balb/c mouse anterior chamber via different concentrations of replicationdefective adenovirus vector serotype 5. Methods Twenty-four Balb/c mice were randomly divided into three equal groups. 2 μL （4 × 10^8 vp/μL） Ad-EGFP was injected into the left eye anterior chamber and 2 μL（4 × 10^7 vp/μL）Ad-EGFP into the right eye anterior chamber in the first group（n = 8）. 2 μL （4 × 10^6vp/μL） Ad-EGFP was injected into the left eye anterior chamber and 2 μL（4× 10^5vp/μL） Ad-EGFP into the right eye anterior chamber in the second group（ n = 8）. The mice of control group received PBS or noninjection（ n = 8）. Fluorescence stereomicroscope was used to observe the expression of EGFP in eye at day 1 , 3,7,10,14 and 25. Confocal microscopy was performed to observe the type and distribution of EGFP positive cells. Results The expression of EGFP was not detected over the ocular surface in the low concentration group （4 × 10^6vp/μL Ad-EGFP and 4× 10^5vp/μL Ad-EGFP） at any time point. Ad5-EGFP was expressed over the ocular surface in the high concentration group （4 × 10^8vp/μL Ad-EGFP and 4 × 10^7vp/μL Ad-EGFP） at day 1. The largest and strongest fluorescence was seen at day 10 and gradually disappeared after that. And the expression of EGFP remained up to 25 days in 4 × 10^8vp/μL Ad-EGFP group. Confocal microscopy results suggested that the EGFP expression was positive in corneal endothelial cells ,iris pigment epithelial cells and trabecular meshwork. Conclusion Replication-defective adenoviral vector serotype 5-mediated report gene could be directionally transducted in ocu

关 键 词：复制缺陷型腺病毒载体 基因治疗 角膜内皮细胞 报告基因 

分 类 号：R77[医药卫生—眼科]