Comparison of transfection efficiency of THP-1 monocytes by different methods

doi:10.3969/j.issn.2095-4344.2014.38.007

Abstract

Abstract:

BACKGROUND: Human THP-1 is a typical suspension cell line. It is more difficult to transfect and transiently express foreign genes into the adherent cells than the suspension cells. Diethylaminoethyl dextran (DEAE-dextran) transfection method has been previously established, but rarely used in China.
OBJECTIVE: To transfect plasmid vector coding enhanced green fluorescent protein N1 into human THP-1 monocytes by different methods and to acquire the method with better transfection efficiency.
METHODS: The cells were transfected by different methods with DEAE-dextran, Lipofectamine 2000, FuGENE 6, Sofast transfection reagents. Transfection efficiency and cell viability were determined.
RESULTS AND CONCLUSION: The transfection efficiency was the highest by DEAE-dextran transfection, then by Lipfectamine 2000, and lowest by FuGENE 6 and Sofast transfection reagent, as detected by fluorescence microscope and flow cytometry. Plasmid enhanced green fluorescent protein N1 was effectively expressed after being transfected into THP-1 monocytes in vitro, and the transfection efficiency was enhanced and the impact on cell viability was reduced by optimizing the transfection methods.

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: monocytes, transfection, plasmids

CLC Number:

R318

Ge Jing, Cheng Bei, Peng Wen, Li Yun-qiao, Zhai Wei . Comparison of transfection efficiency of THP-1 monocytes by different methods[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(38): 6105-6109.

Figures/Tables 1

2.1 DEAE-Dextran对THP-1细胞的毒性影响 DEAE-Dextran对THP-1细胞的毒性与其剂量和作用时间呈正相关性(见图1)，在给予DEAE-Dextran孵育大剂量 (> 600 mg/L)以及各剂量孵育长时间(> 40 min)后，细胞死亡率明显增加达到> 50%。说明THP-1细胞对DEAE- Dextran的耐受性不强，以DEAE-Dextran为介质转染外源质粒，其毒性是不可忽略的因素之一，孵育时间和剂量必须加以严格控制。 2.2 DEAE-Dextran介导的THP-1细胞转染外源质粒的效率优化结果 2.2.1 DEAE-Dextran的剂量及孵育时间的优化 DEAE- Dextran对THP-1细胞转染外源质粒的效率与转染时用的DEAE-Dextran剂量与孵育时间呈正相关(见图2)，在使用 50 mg/L和100 mg/L的DEAE-Dextran时在各个细胞孵育时间转染效率都非常低；在DEAE-Dextran 150 mg/L，各孵时间段都有≥ 5%的转染效率，在孵育时间20和40 min时转染效率较高，为7%-8%；在DEAE-Dextran 300 mg/L，各孵时间段也有> 5%的转染效率，在孵育时间20 min时最高约13%。从以上数据可以看到选用DEAE-Dextran 300 mg/L，20 min可能是比较好的转染条件。 2.2.2 利用不同促进剂 ①二甲基亚砜和甘油：加用甘油组，转染效率较仅用DEAE-Dextran 300 mg/L组无明显升高，约为11%；而二甲基亚砜组不能使转染效率增高，反而有所降低，约为7%。针对THP-1细胞使用二甲基亚砜和甘油并不能增加DEAE-Dextran介导的瞬时转染效率，而且也增加操作步骤，所以不考虑采用这两种促进剂。②氯喹：使用氯喹后，转染效率约为8%，也并不能增加DEAE-Dextran介导的瞬时转染效率，所以也不考虑加用氯喹。 2.2.3 DEAE-Dextran预处理转染方法 DEAE-Dextran预处理转染方法效率并不高，为5%左右，所以也不考虑采用。 2.3 DEAE-Dextran介导的THP-1细胞瞬时转染方法与其他转染方法的比较 2.3.1 荧光显微镜下观察结果采用DEAE-Dextran、Lipofectamine 2000、FuGENE 6、梭华-Sofast 4种不同方法转染THP-1细胞后比较，DEAE-Dextran介导的转染效率最高，Lipfectamine 2000脂质体次之，FuGENE 6和梭华-Sofast则明显低于前二者。 2.3.2 流式细胞仪检测结果于转染48 h后行流式细胞仪检测，结果表明转染效率从高到低依次为DEAE-Dextran、Lipofectamine 2000、FuGENE 6、梭华- Sofast介导的转染，分别为22.40%，9.62%，1.85%，1.01%(见图3)，与阴性对照组比较差异均存在显著性意义(P < 0.05，n=3)，与荧光显微镜观察结果一致。"

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