摘要
目的 观察脂质体是否能介导目的基因转移至视网膜及脂质体潜在的毒副作用 ,探讨脂质体在视网膜疾病基因治疗中的应用价值。方法 通过体外细胞转染实验优化阳离子脂质体与质粒DNA的比例 ,然后将脂质体 DNA混合物注射至Spregue Dawley(SD)及Royalcollegeofsurgeon(RCS)大鼠视网膜下间隙 ,荧光显微镜观察目的基因的表达 ,病理学检查及电镜观察视网膜的组织形态学变化。结果 阳离子脂质体介导转移的绿色荧光蛋白表达质粒在视网膜细胞中表达时间 >4周。但转染后 4周 ,正常SD大鼠的光感受器外节开始出现肿胀变性 ;8周时光感受器的细胞核明显减少 ,仅剩单层细胞 ,外节脱落或消失 ;双极细胞也出现凋亡 ;视网膜内可见新生血管。 5只 4周龄的RCS大鼠经阳离子脂质体介导睫状体神经营养因子 (cillaryneurotrophicfactor,CNTF)表达质粒转移至视网膜 ,3～ 5周时镜下见 3只实验眼视网膜光感受器保存情况明显比对照眼好。结论 阳离子脂质体可有效介导目的基因转移至视网膜细胞内 ;脂质体介导的CNTF基因转移对RCS大鼠视网膜光感受器有一定的保护作用 ,但对正常的视网膜神经细胞特别是光感受器有一定的毒副作用。
Objective To evaluate the efficiency of liposome mediated gene transfer to retina, the time of gene expression in retinal cells, retinal damage caused by liposome and the possibility of using liposome mediated gene transfer to retina for treating fundus diseases. Methods The ratio of cationic liposome to green fluorescein protein (GFP) expression plasmid for high efficacy of transfection was tested in vitro. Then optimal liposome plasmid mixture was injected into the subretinal space of Sprague Dawley (SD) or Royal college of surgeon (RCS) rats. The GFP expression was observed by using fluorescence microscopy, and morphological changes were examined by using paraffin embedded sections, HE staining and transmission electron microscopy. Results The GFP expression in retinal cells was observed lasting more than 4 weeks. However, upon 4 weeks of transfection the swelling and degeneration in outer segment of photoreceptors could be observed. The aggravated degeneration with disappearance of outer segments, only one layer of photoreceptor nucleus surviving, apoptotic inner granular layer cells and neovascularization were observed 8 weeks after transfection. But no inflammatory cells were observed. Three of 5 RCS rats that received plasmid pCNTF (ciliary neurotrophic factor) delivery at 4 weeks old showed more surviving nuclei of photoreceptors after 3 to 5 weeks of transfection. Conclusion Liposome is able to deliver genes to retinal cells with efficacy, and the expression of therapeutic gene such as CNTF in retinal cells can rescue photoreceptors from degeneration. But liposome used here is to some extent harmful to photoreceptors.
出处
《中华眼科杂志》
CAS
CSCD
北大核心
2002年第9期520-522,共3页
Chinese Journal of Ophthalmology
基金
国家自然科学基金资助项目 (3 9770 787)
