In Renal Tubular Epithelial Cells,the Impacts And Mechanisms Of AG1478 On TGF-?1 Inducing The Occurrence Of EMT

Objective: In NRK-52 E and HK-2 cells,investigate the impact of the inhibitor of EGFR,AG1478,in the activation of EGFR and the occurrence epithelial–mesenchymal transition(EMT)induced by TGF-?1.Methods: 1.At the four time points of 0?24?48 and 72 hours,use TGF-?1(10ng/ml)to stimulate NRK-52 E and HK-2 cells respectively,then by WB technology to detect the condition of the expression of epithelial(E-cadherin)and mesenchymal(Vimentin)marker proteins in these two cells;then selecting the most obvious time point of inducing EMT through the results of WB,use TGF-?1(10ng/ml)to stimulate these two cells,then by fluorescent agent to dye the skeleton of cells to see the changes of the cells form.2.At the six time points of 0?0.5?1?3?6 and 24 hours,use TGF-?1(10ng/ml)to stimulate NRk-52 E and HK-2 cells respectively,then by WB technology to detect the condition of the phosphorylation of EGFR in the six time points of these two cells.3.After choosing the most obvious time point of TGF-?1 inducing the phosphorylation of EGFR from method 2,then dealing with these two cells as follows group: Blank Control group,TGF-?1 group,TGF-?1+AG1478(the inhibitor of EGFR)group,AG1478 group,among of this the concentration of TGF-?1 and AG1478 respectively are 10ng/ml,10?M,then through WB by to detect the condition of the phosphorylation of EGFR;4.After choosing the most obvious time point of TGF-?1 inducing EMT from method 1,deal with these two cells as method 3(the concentration of TGF-?1 and AG1478 is unaltered).Then use WB technology to detect the condition of the expression of epithelial and mesenchymal marker proteins;use the fluorescent agent to dye the skeleton of cells to see the changes of form under different treatments simultaneously.Result:1.WB technology detects out the expression of E-cadherin appearing to be decreased at 48 and 72 hours,the Vimentin appearing to be increased at three time points of 24?48 and 72 hours both of these two cells.The results of dye: cells arise to lamellipodia and stress fibers and become to the fusiform shape after stimulated 48 hours.2.WB technology detects out the level of phosphorylation of EGFR appearing to dramatically up-regulation at time point of 3 hours and down-regulation significantly at the time point of 6 hours in these two cells.3.After through the result 2 confirming the most obvious time point of the activation of EGFR(3 hours),the result of WB technology detected as follows,the expression of the level of the EGFR's phosphorylation of TGF-?1 group appears to upregulation compared with Blank Control group.The expression of the level of the EGFR's phosphorylation of TGF-?1+AG1478 group and AG1478 group appear to down-regulation compared with TGF-?1 group.The comparison between other groups have not statistical differences.4.After through the result 1 confirming the most obvious time point of inducing the EMT(48 hours),the result of WB technology detected as follows: the expression of the E-cadherin is decreased and the Vimentin is increased of TGF-?1 group compared with Blank Control group.The expression of the E-cadherin is increased and the Vimentin is decreased of TGF-?1+AG1478 group and AG1478 group compared with TGF-?1 group,the comparison between other groups have not statistical differences.The result of dye: AG1478 can prominently reverse the obvious changes of shape that are lamellipodia and stress fibers stimulated by TGF-?1 alone.Conclusion: In HK-2 and NRk-52 E cells,TGF-?1 can induce the occurrence of EMT and the activation of EGFR.The inhibitor of EGFR,AG1478,can block the activation of EGFR induced by TGF-?1 to achieve the effect of weakening the occurrence of EMT.