脂质体介导转染N2a细胞的优化方法.docx

脂质体介导转染N2a细胞的优化方法赵云鹤1，王若楠2，杨桂姣1，陆利11山西医科大学人体解剖学教研室，山西省太原市030001；2山西医科大学七年制临床医学系，山西省太原市030001Optimization of N2a cell transfection mediated by liposomeZhao Yun-he1, Wang Ruo-nan2, Yang Gui-jiao1, Lu Li11Department of Anatomy, Shanxi Medical University, Taiyuan 030001, Shanxi Province, China; 2Seven-year Program of Clinical Medicine, Shanxi Medical University, Taiyuan 030001, Shanxi Province, China摘要 背景：阳离子脂质体介导的细胞转染具有结果可靠、可重复性强的特点，但面临一个共同的缺点，就是转染效率常较低。目的：探讨阳离子脂质体转染N2a细胞(小鼠神经胶质瘤细胞)的优化方法。方法：采用24孔培养板，1.5 L阳离子脂质体Lipofectamine LTX介导，通过贴壁法和悬浮法，将500 ng带有绿色荧光蛋白(GFP)基因的重组质粒pcDNA3-GFP转入N2a细胞，倒置荧光显微镜观察细胞内绿色荧光蛋白表达情况，并比较二者转染效率。采用悬浮转染法，将500 ng质粒DNA分别用1.0，1.5，2.0，2.5 L Lipofectamine LTX进行转染，探讨最适脂质体和DNA比例。结果与结论：1.5 L脂质体/500 ng DNA，悬浮法转染效率显著高于贴壁法转染效率(P 0.01)；1.0，1.5，2.0，2.5 L Lipofectamine LTX对500 ng质粒DNA的转染效率分别为(76.603.85)%，(80.004.17)%，(88.005.89)%，(54.964.23)%，提示脂质体2.0 L与质粒DNA 500 ng的转染效率最高。中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程全文链接：窗体顶端关键词 ： 组织构建； 组织工程； 脂质体； 贴壁转染； 悬浮转染； N2a细胞； 绿色荧光蛋白； Abstract： BACKGROUND: Cationic liposome-mediated cell transfection is reliable and repeatable. However the transfection efficiency is often low.OBJECTIVE: To study the optimized methods for gene transfection mediated by liposome into N2a cells (mouse neuroblastma cells).METHODS: Using traditional adherent method and improved suspension method, 500 ng recombinant plasmid pcDNA3-GFP carrying green fluorescence protein was transfected into N2a cells in 24-well culture plate, which was mediated by 1.5 L Lipofectamine LTX Reagent. The expression of green fluorescent protein was observed by inverted fluorescence microscope, and the transfection efficiencies at different transfection ways were calculated. By using improved suspension transfection method, 500 ng plasmid DNA was transfected with different doses of Lipofectamine LTX Reagent (1.0, 1.5, 2.0, 2.5 L). The optimal ratio of liposome and DNA was explored.RESULTS AND CONCLUSION: The transfection efficiency of suspension transfection method was significantly higher than that of the tranditional adherent method (P 0.01) when using 1.5 L liposome/500 ng DNA. The transfection efficiency of the 1.0, 1.5, 2.0, 2.5 L Lipofectamine LTX on 500 ng plasmid DNA was respectively (76.603.85)%, (80.004.17)%, (88.005.89)%, (54.964.23)%. It showed the 500 ng DNA and 2.0 L liposome achieve the highest transfection efficiency.中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程全文链接：Key words： liposome; transfection; green fluorescent protein; 窗体底端 中图分类号:R318 基金资助: 山西省研究生创新基金项目(20133075)；山西省高等学校大学生创新创业培训项目(2013119)；山西医科大学青年科技研究基金项目(02201101)通讯作者: 陆利，博士，教授，硕士生导师，山西医科大学人体解剖学教研室，山西省太原市 030001 作者简介: 赵云鹤，女，1981年生，山西省阳泉市人，山西医科大学在读博士，主要从事神经干细胞衰老机制的相关研究。引用本文: 赵云鹤,王若楠,杨桂姣等. 脂质体介导转染N2a细胞的优化方法J. 中国组织工程研究, 2014, 18(29): 4669-4674. Zhao Yun-he,Wang Ruo-nan,Yang Gu