动物造模 z-bio 2022-05-10 568

　　Objective To screen and verify the conditions of electroporation for transfection of TRIB3 overexpression plasmid and TRIB3 siRNA in RAW264.7 cells, and to establish the optimal electroporation transfection scheme for RAW264.7 cells.

　　Methods By changing the pulse voltage, pulse duration and plasmid concentration, the TRIB3 overexpression plasmid and TRIB3 siRNA with the vector pCMV6-Entry were transferred into RAW264.7 cells. After 24 hours, the cell morphology was observed by phase contrast microscope, and the cells were detected by CCK-8 method. Survival rate, TRIB3 protein expression was detected by Western Blot after 72 h to verify the change of target gene expression.

　　Results (1) When transfected with different pulse voltages, the morphology and viability of cells in the 110 and 120 mV groups were not different from those in the control group, while the 130 and 140 mV groups had more suspended cells and the cell viability was significantly lower than that in the control group (P<0 . 01), 120, 130 and 140 mV groups showed a significant increase in TRIB3 protein expression (P < 0.05); (2) when transfected with different pulse durations, the cell morphology and viability of the 10 and 20 ms groups were no different from those of the control group. The cell viability in the 30 ms group was significantly lower than that in the control group (P < 0. 01), and the expression of TRIB3 protein in the 20 and 30 ms groups was significantly increased (P < 0. 01); (3) when transfected with different plasmid concentrations , 2 and 4 μg groups had no difference in cell morphology and viability compared with the control group, the 6 μg group had lower cell viability than the control group (P<0.05), and the 4 μg group had a significant increase in the expression of TRIB3 (P<0.01); (4) When transfected with different concentrations of TRIB3 siRNA at 120 mV and 20 ms, the morphology and survival rate of cells in each group were no different from those in the control group, and the expression of TRIB3 in the 10 nmol/L group was only 0.19 times that of the control group (P < 0.01).

　　Conclusion Electroporation of TRIB3 overexpression plasmid (4 μg per sample) and TRIB3 siRNA (10 nmol/L) in RAW264. 7 cells under the conditions of 120 mV and 20 ms can significantly improve cell viability while ensuring high cell viability. Change the protein expression level of the target gene, and provide experimental basis and reference for subsequent basic research on plasmid transfection in RAW264.7 cells.