巨噬细胞RAW264.7的培养及电转染条件的优化  

Characterization of Macrophage Culture and Optimization of Electric-mediated Transfection

作　　者：宋永利[1,2] 陆昕 邱爽[2] 祁英培[2] 华松[2] 兰杰[2] 何小宁[2] 贺小英[2] 张涌[2] 郑月茂[2] 

机构地区：[1]吉林农业科技学院动物医学院,吉林132101 [2]西北农林科技大学动物医学院,农业部动物生殖生理与胚胎工程重点开放实验室,陕西杨凌712100 [3]西安市雁塔区动物疾病预防中心,西安710061

出　　处：《西北农业学报》2010年第9期21-24,共4页Acta Agriculturae Boreali-occidentalis Sinica

基　　金：抗病转基因牛新品种培育(2008ZX08007-004)

摘　　要：为了建立最优的巨噬细胞RAW264.7培养方法和电转染条件,在细胞培养时采用两种方法了解细胞的培养特性并进行电转染条件的优化,建立并验证RAW264.7细胞快速、无血清培养体系的电转染方法。以绿色荧光蛋白（GFP）为报告基因,进一步优化巨噬细胞电穿孔的条件。通过控制电压（250~420 V）、脉冲时间、电击次数、温度、电击缓冲液等转染条件,采用不同的条件组合,用电穿孔法将质粒pEGFP-C1转染于培养的RAW 264.7细胞。通过荧光显微镜分析转染效率,可见：用胰酶和细胞刮刀消化RAW264.7细胞,细胞损伤小,形态好。利用优化的电转染条件得出最佳转染条件是：电压350 V、脉冲时间30 ms、电击1次、电击缓冲液成分为cell盐和options且V（cell盐）∶V（options）=3∶1、最佳温度4℃。This paper intended establishing the optimal methods for cell culture and electric transfection based on macrophage RAW264.7.For electric transfection,parameters involving voltage,impulsion time,electronic shock times,temperature and shock buffer solution were investigated.Cultured RAW264.7 cells were transfected with pEGFP-C1 by using GFP as an indicator depending on different combinations of parameters.The results showed that trypsin digestion combined with scraper were better,with the less damaged cell.The optimal conditions of electric transfection were 350 volt in voltage,30 ms for impulsion time,1 time for electronic shock,the ratio of 3∶1 for shock buffer solution to cell salt,as well as 4 degree Celsius for temperature.

关 键 词：巨噬细胞 体外培养 基因转染 

分 类 号：Q813[生物学—生物工程]