摘要
试验旨在比较不同转染方法介导绿色荧光蛋白(EGFP)基因转染猪不同体细胞的效果。原代分离培养来源于猪脂肪和骨髓的两种间充质干细及成纤维细胞,含有EGFP的质粒分别通过脂质体、罗氏转染试剂和电穿孔法转入猪3种体细胞中,使用倒置荧光显微镜观察EGFP的表达,并通过血细胞计数法分析细胞相对存活率,流式细胞仪检测细胞转染效率。结果表明,3种方法转染不同体细胞48 h均有绿色荧光蛋白的表达。在不同方法转染相同体细胞的比较中,细胞存活率从高到低依次为罗氏转染试剂>脂质体>电穿孔法,罗氏转染试剂和脂质体转染后的细胞存活率显著高于电穿孔法(P<0.05),转染效率从高到低依次为电穿孔法>罗氏转染试剂>脂质体,电穿孔法的转染效率明显高于罗氏和脂质体转染试剂,同时罗氏转染试剂的转染效率显著高于脂质体(P<0.05)。采用相同方法转染不同体细胞的比较中,3种体细胞在相同方法转染后的存活率无显著性差异(P>0.05),电穿孔法转染间充质干细胞的效率均显著高于成纤维细胞(P<0.05);罗氏转染试剂和脂质体分别转染3种体细胞的效率无显著性差异(P>0.05)。3种方法均可用于转染猪体细胞,综合考虑转染效率和细胞存活率,电穿孔法的转染效果最好,可用于转染细胞状态较佳的细胞,其次为罗氏转染试剂。
The objective of this paper is to compare the effect of different transfection methods mediating the transfection of enhanced green fluorescent protein(EGFP)gene into different porcine somatic cells.Porcine adipose and bone marrow-derived mesenchymal stem cells(MSCs)and porcine fetal fibroblasts were isolated and cultured.The plasmid containing EGFP was transfected into each of the three cell types using electroporation,liposome and Roche transfection reagent,respectively.Fluorescence expression was observed by inverted fluorescence microscopy,and relative cell viability and transfection efficiency were analyzed by blood count and flow cytometry.The results showed that EGFP expression was observed in all three methods of transfection of different somatic cells for 48 hours.In the comparison of different methods of transfecting the same somatic cells,the cell survival rate was in the order of Roche transfection reagent>liposome>electroporation method,the survival rate of cells transfected with Roche transfection reagent and liposome was significantly higher than that of electroporation(P<0.05).The transfection efficiency in descending order was electroporation>Roche transfection reagent>liposome,with electroporation significantly higher than Roche transfection reagent and liposome,the transfection efficiency of Roche transfection reagent was also significantly higher than that of liposome(P<0.05).In the comparison of transfection of different somatic cells using the same method,there was no significant difference in the survival rate of the three somatic cells after transfection by the same method(P>0.05),and the transfection efficiency of MSCs by electroporation were significantly higher than the porcine fetal fibroblasts(P<0.05).There was no significant difference in transfection efficiency between the three types of somatic cells transfected with Roche transfection reagent and liposomes respectively(P>0.05).All three methods can be used to transfect porcine somatic cells.Considering transfection efficiency and
作者
赵广伟
杨小玲
张孟奇
苏勤智
陈凤
ZHAO Guangwei;YANG Xiaoling;ZHANG Mengqi;SU Qinzhi;CHEN Feng(College of Animal Science and Technology,Henan University of Animal Husbandry and Economy,Zhengzhou 450046,Henan;Digestive Medicine Center,The Seventh Affiliated Hospital of Sun Yat-sen University,Shenzhen 518107,Guangdong)
出处
《家畜生态学报》
北大核心
2024年第2期13-18,共6页
Journal of Domestic Animal Ecology
基金
河南牧业经济学院博士科研启动基金项目(24030028)。
关键词
电穿孔法
脂质体
罗氏转染试剂
体细胞
转染效率
细胞存活率
electroporation
liposome
Roche transfection reagent
somatic cells
transfection efficiency
cell viability
