脂质体介导转染犬肾上皮细胞的方法改进与优化  

Improvement and Optimization of Liposome-Mediated Transfection of Madin-Darby Canine Kidney Cells

作　　者：柴百惠 绳秀珍 唐小千 邢婧 战文斌[1] CHAI Bai-Hui;SHENG Xiu-Zhen;TANG Xiao-Qian;XING Jing;ZHAN Wen-Bin(The Key Laboratory of Mariculture(Ocean University of China),Ministry of Education,Qingdao 266003,China)

机构地区：[1]海水养殖教育部重点实验室(中国海洋大学),山东青岛266003

出　　处：《中国海洋大学学报（自然科学版）》2018年第A02期9-16,共8页Periodical of Ocean University of China

基　　金：国家自然科学基金项目(31472295;31730101;31672685);山东省泰山学者特聘专家项目资助~~

摘　　要：本文使用带绿色荧光蛋白报告基因的真核质粒pCI-neo-EGFP作为外源DNA,将脂质体介导转染犬肾上皮细胞(MDCK)的常规转染法改进为悬浮法,并比较了二者在转染36h后的转染效果,结果显示悬浮法的转染效率显著优于常规法。研究了悬浮转染法不同DNA用量、脂质体/DNA比例和悬浮孵育时间对转染效率和细胞活力的影响。流式细胞术检测结果显示,转染效率与DNA用量呈正相关依赖关系;在DNA用量相同时,转染效率随着脂质体/DNA比例升高呈先上升后下降的趋势;DNA用量为1μg、脂质体/DNA比例为4∶1、悬浮孵育时间为20 min时转染效率最高(21.07%±0.76%)。利用CCK-8细胞毒性试剂盒测定不同条件下的细胞活力,结果表明细胞活力随DNA用量和脂质体/DNA比例的升高及悬浮孵育时间的延长而降低,当DNA用量(0.25μg)和脂质体/DNA比例(2∶1)最低、悬浮孵育20min时,细胞活力最高(95.67%±3.72%),而转染效率最高时细胞活力为69.95%±3.01%。研究结果为进一步建立稳定转染细胞研究模型提供了基础资料。In this study,the conventional method of liposome-mediated transfection of Madin-Darby canine kidney cell lines(MDCK)was modified to suspension method using the eukaryotic plasmid pCI-neo-EGFP containing reporter gene,green fluorescent protein(GFP)encoding gene,as the exogenous DNA.After transfection for 36 h,transfection efficiency of the improved transfection method was significantly higher than that of the conventional one.The effects of different amounts of DNA,liposome/DNA ratio and suspension incubation time on the transfection efficiency and cell viability were studied.Flow cytometry analysis indicated that the transfection efficiency was positively correlated with the amount of DNA.When the amount of DNA was the same,the transfection efficiency increased first and then decreased with the increase of the liposome/DNA ratio.The highest transfection efficiency(21.07%±0.76%)was obtained when the DNA dosage was 1μg,the liposome/DNA ratio was 4:1 and the suspension incubation time was 20 min.The viability of MDCK cells under different conditions was determined using the CCK-8 cytotoxicity kit.It was found that the cell viability decreased with the increase of DNA amount,liposome/DNA ratio and the suspension incubation time.When the amount of DNA,liposome/DNA ratio and suspension incubation time were the lowest(0.25μg,2∶1 and 20 min,respectively),the cell viability was the highest(95.67%±3.72%).The cell viability was 69.95%±3.01%if the transfection efficiency was the highest.These results provided basic data for further establishment of a stably transfected cell model.

关 键 词：脂质体 瞬时转染 犬肾上皮连续细胞系(MDCK) 悬浮法 转染效率 细胞活力 

分 类 号：S917.4[农业科学—水产科学] S942