Transfection Efficiency And Cytotoxicity Comparison Of Different Transfection Reagent On Transfecting RIP140-siRNA To Kupffer Cells

Objective: To compare the efficiency and cytotoxicity of different transfection reagents transfecting RIP140-si RNA to kupffer cells, so as to find out the best reagent transfection methods and conditions.Methods: Kupffer cells as the research object, RIP140-si RNA marked with GFP as the reporter gene, the lipofectamine 2000, roche Reagent(X – treme GENE si RNA Transfection Reagent) and puro screening lentivirus(1.0×108TU/ml) as Transfection reagents, using fluorescent inverted microscope to observe the cell transfection effect, using ELISA kit to detect the expression of NF-κb,IL-2,IL-10,TNF-α and analyze the change of its immune activity, laser scanning confocal microscope to analysis the expression of RIP140 of kupffer cells after transfection, Flow cytometry to detect each cell apoptosis, CCK-8 testing each cell proliferation inhibition. Collecting cell and cracking, extract RNA and protein, using RT-RCR and Western blotting experiment method to detect the gene and protein expression of RIP140 of kupffer cells after transfection,P < 0.05 for the difference was statistically significant.Results: To kupffer cells, in the aspect of transfection efficiency, the transfection efficiency of puro screening lentivirus is the highest, which can reach more than 90% percent; Roche reagent is the secondly; the transfection efficiency of lipofectamine 2000 is the worst. In terms of reagent cytotoxicity, the test results of flow cytometry and CCK-8 show that roche reagent cytotoxicityis the smallest, the cells keep their original form; Lentivirus is the secondly; The cytotoxicity of lipofectamine 2000 is the largest, most visible cells lose their original shape,which at the state of cell lysis. ELISA results show that the immunocompetence of lentivirus group(P<0.05) has biggest change, the secretion of NF-κb,IL-2,IL-10,TNF- α appeared different degree of increase or decrease after the transfection peak, difference between the other two groups is not obvious(P>0.05). RT-RCR and Western blotting experiments showed that no matter in terms of gene or protein levels, the lentivirus transfection cell group were significantly lower than lipofectamine 2000 and roche reagent transfection cell group(P<0.05).Conclusions: For kupffer cells, in terms of reagent transfection, lentivirus transfection method can achieve ideal transfection efficiency and smaller cytotoxicity, and the more superior conditions controllability and stability.