阳离子聚合体介导下角膜基质注射GFP基因及其表达  

Expression of plasmid DNA encoding GFP mediated by cationic polymer after its intrastromal injection

作　　者：袁进[1] 陈家祺[1] 周世有[1] 陈海[2] 

机构地区：[1]中山大学中山眼科中心,广州510060 [2]中山大学生命科学院生化实验室

出　　处：《眼科研究》2005年第3期225-227,共3页Chinese Ophthalmic Research

基　　金：国家自然科学基金资助(30271388)

摘　　要：目的研究角膜层间注射阳离子聚合体/质粒复合物转染角膜组织的有效性和安全性。方法用微量进样器将表达绿色荧光蛋白(GFP)的重组质粒PEGFPC2和阳离子聚合体的混合溶液16μL注射于Wistar大鼠角膜基质,对照组注射等量NS。注射后不同时间点(1、3、14、21d)收集角膜行HE染色、透射电镜和荧光显微镜检查结果电镜证实角膜细胞内可见PEI/DNA颗粒。HE染色未见炎症细胞浸润。实验组角膜1d后GFP开始表达,3d时达到高峰,全角膜均表达阳性,21d时仍有微弱表达。对照组角膜未见荧光表达。结论角膜基质注射阳离子复合体包装的质粒可将外源性基因安全地、相对高效地转入角膜组织。ObjectiveTo investigate the efficiency and safety of injection of plasmid DNA encoding green fluorescent protein (GFP) into corneal stroma.MethodsCationic polymer (polyethylenimine,PEI) and plasmid encoding GFP complex were prepared under microscope and 16μL PEI/DNA complex was directly injected into the corneal stroma of Wistar rat.Normal sodium was injected into cornea in the control group.The corneas were harvested at day 1,3,14,and 21 after injection for the structure analysis of cornea by HE staining and transmission electron microscope (TEM).Expression of the transferred gene was monitored by fluorescence microscopy.ResultsPEI/DNA complex solution dispersed in the cornea and lead to corneal slight edema but became transparent after 1 day.No inflammation and neovascularization were developed in the cornea.The granule of PEI/DNA complex were seen in keratocytes,and no significant toxicity was detected under the TEM.The fluorescence intensity of expression of GFP in the cornea was increasing with time and pecked on days 3 and declined afterward.GFP was absent staining in the control group.ConclusionForeign plasmid DNA mediated by cationic polymer could be transferred into keratocyte by directly injecting into corneal stroma.It may provide a new technique of gene transfer into cornea in situ.

关 键 词：角膜 基因转染 绿色荧光蛋白 阳离子聚合体 

分 类 号：R346[医药卫生—基础医学]