重组腺相关病毒介导增强型绿色荧光蛋白基因经角膜基质瓣转染兔角膜基质细胞的实验研究  

Experimental study of recombinant adeno-associated virus-mediated enhanced green fluorescent protein gene transfection of rabbit keratocytes with a lamellar flap

作　　者：汤明芳[1] 陆晓和[2] 马骊[3] 温茜[3] 罗微[3] 周瑾[2] 袁伟[2] 宫玉波[2] 

机构地区：[1]南方医科大学南方医院眼科,广州510515 [2]南方医科大学珠江医院眼科,广州510282 [3]南方医科大学生物技术学院分子免疫学研究所,广州510515

出　　处：《广东医学》2008年第4期542-544,共3页Guangdong Medical Journal

基　　金：广东省自然科学基金资助项目(编号:0402438)

摘　　要：目的研究重组腺相关病毒(rAAV)介导增强型绿色荧光蛋白(EGFP)基因经角膜基质瓣转染兔角膜基质细胞的有效性和安全性。方法微型角膜刀制作兔角膜基质瓣;转染组用含rAAV-EGFP转染液浸泡角膜瓣和角膜基质床10min,对照组用等量BSS液浸泡角膜瓣和角膜基质床10min,不同时间点(1,3,7,14,21d)通过荧光体视镜观察角膜出现EGFP荧光的起始时间及分布情况,收集角膜,其中一半用激光共聚焦显微镜观察、照相,另一半固定后行HE染色。结果转染组于转染第3天,体视荧光显微镜下观察到兔眼角膜瓣内面及基质床中开始有GFP阳性表达;7d达到高峰,21d时仍有微弱表达。第7天在激光共聚焦显微镜下可观察到兔角膜基质中有明显的荧光表达,对照组角膜始终未见荧光表达,转染组和对照组角膜HE染色均未见炎症细胞浸润及新生血管等异常。结论rAAV-EGFP经兔角膜基质瓣转染角膜基质细胞的方法可将基因安全、相对高效地转入角膜基质细胞,为转基因治疗角膜病提供了新的转染途径。Objective To investigate the effciency and safety of recombinant adeno - associated virus （rAAV） - mediated enhanced green fluorescent protein gene （EGFP） transfection in rabbit keratocytes with a lamellar flap. Methods A lamellar flap was produced in rabbit cornea with a microkeratome. The flap interface in whole corneas and corneal beds were soaked in the rAAV - EGFP solution for 10 min. BSS solution was used in the control group. The corneas were harvested at day 1,3, 14, and 21 after treatment for morphological analysis by HE staining. Expression of the transferred gene was monitored by fluorescence stereoscope and laser confocal microscope. Results After 3 d of infection with rAAV -EGFP,slight fluorescence was detected in rabbit cornea by fluorescence stereoscope. GFP continued to be expressed in rabbit cornea. Its expression in rabbit keratocytes increased with time and reached maximum in 7 d and declined after- ward. Significant expression of GFP in the rabbit corneal stroma was detected by laser confocal microscope in 7 d. GFP staining was absent in the control group. No inflammation and neovascularization were developed in the cornea. Conclusion rAAV - EGFP mediated gene transfer in rabbit cornea using a lamellar flap - technique can be transferred into corneal keratocytes. A safe and effeient method of gene transfer into cornea stroma in situ can be developed to provide new treatment for corneal diseases.

关 键 词：角膜 基因转染 绿色荧光蛋白 腺相关病毒 基质辫 

分 类 号：R456[医药卫生—治疗学] R746.2[医药卫生—临床医学]