Comparison of efficiency and cytotoxicity of different transfection reagents in transfecting RIP140-siRNA into Kupffer cells
10.3969/j.issn.1673-4254.2015.12.06
- VernacularTitle:不同试剂转染RIP140-siRNA至库普弗细胞的效率及毒性比较
- Author:
Ji LI
1
;
Zuojin LIU
Author Information
1. 重庆医科大学第二临床学院
- Keywords:
liver kupffer cell;
transfection efficiency;
cytotoxicity;
RIP140-siRNA
- From:
Journal of Southern Medical University
2015;(12):1694-1700
- CountryChina
- Language:Chinese
-
Abstract:
Objective To compare the efficiency and cytotoxicity of different transfection reagents used in transfection of RIP140-siRNA into Kupffer cells to optimize the transfection conditions. Methods Kupffer cells were transfected with RIP140-siRNA labeled with GFP as the reporter gene using lipofectamine 2000, Roche reagent (X-treme GENE siRNA Transfection Reagent) and puro screening lentivirus (1.0 × 108 TU/mL) as the transfection reagents. The transfection effect was observed under a fluorescent inverted microscope, and laser scanning confocal microscopy was used to analyze RIP140 expression in trasnfected Kupffer cells. Flow cytometry was performed to detect cell apoptosis, and CCK-8 test was used to evaluate the cell proliferation inhibition. RT-RCR and Western blotting were performed to detect the expressions of RIP140 mRNA and protein in the trasnfected cells. Results Puro screening lentivirus yielded the highest cell transfection efficiency, which exceeded 90%, followed by Roche reagent and then by lipofectamine 2000. Flow cytometry and CCK-8 test showed that the cytotoxicity was the mildest with Roche reagent, moderate with lentivirus, and severe with lipofectamine 2000. The cells trasnfected with lentivirus showed a significantly lower RIP140 expression than cells trasnfected with lipofectamine 2000 and Roche reagent (P<0.05). Conclusion In Kupffer cells, lentivirus-mediated transfection, as compared with the other two trasnfection reagents, can achieve good transfection efficiency with a relativelty low cytotoxicity, and allows for better controllability and stability of the trasnfectiion conditions.
