摘要
目的观察阳离子脂质体Lipofectamine2000能否介导目的基因转移至兔角膜内皮细胞内及脂质体潜在的毒副作用。方法RT-PCR检测特异性胶原Ⅷ进行细胞鉴定,通过体外细胞转染实验优化阳离子脂质体与质粒DNA的浓度和比例,选择Lipo-fectamineTM2000/pEGFP-N1比值3∶1,脂质体体积分别为0μL、6μL、9μL、12μL转染兔角膜内皮细胞,荧光显微镜观察目的基因的表达,透射电镜观察细胞超微结构。结果RT-PCR扩增得到单一产物,与预期的扩增序列大小完全一致,角膜内皮细胞内可见脂质体介导的绿色荧光蛋白表达,LipofectamineTM2000浓度能显著影响其对兔角膜内皮细胞的转染效率,存在最佳浓度,在35mm培养皿中,3μgDNA与9μL脂质体转染效率最高,透射电镜显示12μL脂质体可导致细胞器出现肿胀、崩解现象。结论阳离子脂质体可有效介导目的基因转移至角膜内皮细胞内,在35mm培养皿中,12μL脂质体对角膜内皮细胞有一定的毒副作用。
Objective To evaluate the efficiency of liposome mediated gene transfer into corneal endothelial cells and it＇ s side effect. Methods Cells were identified by RT-PCR detection of mRNA for specific marker-collagen Ⅷ, the ratio of cationic liposome to green fluorescein protein （GFP） expression plasmid for high efficiency of transfection was tested in vitro. Lipofectamine^TM with 0 μL,6 μL,9 μL,12 μL 2000 was mixed to make Lipofectamine^TM 2000/pEGFP-N1 complex, and the ratio of Lipofectamine^TM 2000 to pEGFP-N1 was 3 ： 1. The GFP expression was observed by fluorescence microscopy, the ultrastructure of corneal endothelial cells was observed by transmission electron microscopy. Results RT-PCR verified that cells obtained from the explant cultures of rabbit corneal tissue were corneal endothelial cell, the GFP expression in corneal endothelial cells was observed, the transfection efficiency of GFP was various dependent on the concentration of Lipofectamine^TM 2000. In 35 mm cell cultured plate ,3μg DNA with 9 μL liposome resulted in the highest transfection efficiency.The corneal endothelial cells incubated with 12 μL Lipofectamine^TM 2000,vacuole formation could be seen by transmission electron microscopy. Conclusion Liposome is able to transfer gene into corneal endothelial cells with efficiency, but high concentration of liposome used here is to some extent harmful to corneal endothelial cell.
出处
《眼科新进展》
CAS
2007年第7期485-488,共4页
Recent Advances in Ophthalmology
基金
湖北省自然科学基金资助(编号:2003ABA150)~~
关键词
角膜内皮细胞
脂质体
转染
corneal endothelial cell
liposomes
transfection
