| Object:This study used cytology experiments to select Caco2 cells and stably transfect NHERF4.PI3 K /AKT signaling agonists and inhibitors were used to up-regulate and down-regulate PI3K/AKT signaling pathways to understand NHERF4 expression and intracellular localization.The effect of PI3 K /AKT-SLC26A3,so as to analyze the effect of NHERF4 on the expression of SLC26A3 in UC and lay the foundation for cytology experiments.Methods:1.Caco-2 cells were infected with lentivirus LV-NHERF4.After puromycin sieving,Caco-2 cell lines stably transfected with NHERF4 were obtained.2.Caco-2 cells that were not transfected with NHERF4 and Caco-2 cells that were transfected with NHERF4 plasmid were administered with different concentrations of IGF-1 reagents,and the drug concentration was 0.1ug / m L,0.2ug / m L,0.4ug / m L in order.,1ug / m L,2ug / m L,3ug / m L,observe the expression of NHERF4 and SLC26A3 protein,and analyze the effect of NHERF4 on the effect of PI3 K /AKT-SLC26A3.3.Caco-2 cells that were not transfected with NHERF4 and Caco-2 cells that were transfected with NHERF4 plasmid were administered with different concentrations of LY294002 reagent,respectively,and the drug concentration was 1u M,2u M,3u M,4u M,5u M,6u M.The expression of NHERF4 and SLC26A3 protein was observed,and the effect of NHERF4 on the effect of PI3K/AKT-SLC26A3 was analyzed.results:1.Caco-2 cell line stably transfected with NHERF4 was successfully constructed.The difference between the blank group and the transfection group was statistically significant.2.In Caco-2 cells not transfected with NHERF4,when the PI3K/AKT signaling pathway is activated(N:0.336 ± 0.007,0.1: 0.478 ± 0.001,0.2: 0.488 ± 0.002,0.4: 0.678 ± 0.002,1: 0.613 ± 0.001,2: 0.546 ±0.001,3: 0.455 ± 0.004,P <0.05),NHERF4 was not expressed in the non-transfected group,and the expression of SLC26A3 protein increased with the increase of IGF-1 concentration,increasing to 0.4ug /m L At the peak,when the concentration of IGF-1 was greater than 0.4ug / m L,the expression of SLC26A3 protein decreased(N: 0.427 ± 0.011,0.1: 0.613 ± 0.003,0.2: 0.676 ± 0.001,0.4: 0.976 ± 0.001,1: 0.827 ±0.001,2: 0.729 ± 0.002,3: 0.497 ± 0.001,P <0.05).The expression of SLC26A3 protein in Caco-2 cells transfected with NHERF4 plasmid increased with the increase of IGF-1 concentration,increasing to 0.4ug /It reached a peak at m L.When the concentration of IGF-1 was greater than 0.4ug / m L,the expression of SLC26A3 protein showed a downward trend(N: 0.484 ± 0.002,Z: 0.678 ± 0.009,0.1: 0.794 ± 0.003,0.2:0.798 ± 0.003,0.4 : 0.937 ± 0.006,1: 0.770 ± 0.002,2: 0.786 ± 0.002,3: 0.511 ± 0.007,P <0.05).3.In Caco-2 cells not transfected with NHERF4,when PI3K/AKT signaling pathway is inhibited(N: 0.758± 0.001,1: 0.988 ± 0.007,2: 0.808 ± 0.004,3: 0.684 ± 0.002,4: 0.497 ± 0.005,5: 0.490 ± 0.009,6: 0.465± 0.003,P <0.05),NHERF4 is not expressed,and the expression of SLC26A3 decreases with the increase of LY294002 concentration(N: 0.605 ± 0.001,1: 0.787 ± 0.005,2: 0.958 ± 0.002,3: 0.698 ± 0.001,4:0.756 ± 0.002,5: 0.629 ± 0.007,6: 0.598 ± 0.003,P <0.05).The expression of NHERF4 protein in Caco-2cells transfected with NHERF4 plasmid did not follow the concentration of LY294002.The concentration of SLC26A3 protein decreased as the concentration of LY294002 increased(N: 0.678 ± 0.007,Z: 0.565 ±0.002,1: 0.673 ± 0.007,2: 0.690 ± 0.007,3: 0.612 ± 0.014,4: 0.563 ± 0.003,5: 0.509 ± 0.001,6: 0.510 ±0.005,P <0.05).in conclusion:1.The PI3K/AKT signaling pathway may not affect the expression of NHERF4 in Caco-2 cells.2.This study may suggest that persistently overactivated PI3K/AKT signaling in the intestinal mucosa of patients with ulcerative colitis may be one of the mechanisms of decreased expression of SLC26A3 protein in the intestinal mucosa of patients. |
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