To Investigate Impact About The Active Vitamin D3and Its Receptor On MTOR Protein Expressed In Human Mesangial Cells

Object：To investigate the effect of1,25(OH)2D3and its receptor on cultured humanmesangial cell proliferation,cell cycle,cell apoptosis and its effect on the expression of mTORin human mesangial cell.To further explore2the regulation effect of1,25(OH)2D3and itsreceptor through the PI3K/Akt/mTOR signaling pathway to human mesangial cell.Methods:(1)The cultured human glomerular mesangial cell were divided into thenormal control group,EGF(10ng/ml)group,1,25(OH)2D3(10-8mol/L)group and the EGFcombine1,25(OH)2D3.The cultured human glomerular mesangial cell were treated bydifferent drug for48hours.The morphology and growth of human glomerular mesangial cellwere observed by the inverted phase contrast microscope.(2)The proliferation of humanglomerular mesangial cell were measured by the cell counting kit-8.(3)The cell cycle ofhuman glomerular mesangial cell were measured by the flow cytometry.(4)The apoptosis ofhuman glomerular mesangial cell were measured by the the TdT-mediated dUTP nick-end labeling(TUNEL) assay.(5)The expression of mTORmRNA、nVDRmRNA in each group weremeasured by the reverse transcription PCR.(6)The expression of mTOR and VDR in each groupwere measured by the western-blot.Results:(1)Under the inverted phase contrast microscope, The human glomerular mesangialcell of the normal control group assumes the spindle,the cell nucleus was in the middle of thecell,the cell nucleus was round or ovoid.Compared with the normal control group,the humanglomerular mesangial cell of the EGF group grow faster.the cell volume was increased,evenshowing binucleated cells. the human glomerular mesangial cell of the1,25(OH)2D3groupwas significantly inhibited. Characterized by cell shrinkage, smaller size,condensed nucleusand cytoplasm even vacuoles.the human glomerular mesangial cell of the EGF combine1,25(OH)2D3group was poor growth.(2)The result of the cell counting kit-8showed:Compared with the normal control group,the EGF group was significantly promotethe proliferation of human glomerular mesangial cell,the difference was statisticallysignificant(P＜0.01);the1,25(OH)2D3group was significantly inhibited,the difference wasstatistically significant(P＜0.01);Compared with the EGF group,the EGF combine 1,25(OH)2D3group can significantly inhibit the proliferation of human glomerular mesangialcells,the difference was statistically significant(P＜0.01).(2)The result of the cell cycleshowed:Compared with the normal control group,The cells of G1phase in EGF group weresignificantly decreased and the cells of S phase were significantly increased,the difference wasstatistically significant(P＜0.01);The cells of G1phase in1,25(OH)2D3group weresignificantly increased and the cells of S phase were significantly decreased, the difference wasstatistically significant(P＜0.01).Compared with the EGF group,The cells of G1phase in EGFcombine1,25(OH)2D3group were significantly increased and the cells of S phase weresignificantly decreased, the difference was statistically significant(P＜0.01).(4)The result ofcell apoptosis showed:Compared with the normal control group,the cell apoptosis rate of theEGF group was significantly decreased,the difference was statistically significant(P＜0.01).the cell apoptosis rate of the1,25(OH)2D3group was significantly increased,thedifference was statistically significant(P＜0.01).Compared with the EGF group,the cellapoptosis rate of the EGF combine1,25(OH)2D3group was significantly increased,thedifference was statistically significant(P＜0.01).(5)the result of mTORmRNAshowed:Compared with the normal control group,the expression of mTORmRNA in EGFgroup was significantly increased,the difference was statistically significant(P＜0.01),theexpression of mTORmRNA in1,25(OH)2D3group was significantly decreased,the differencewas statistically significant(P＜0.01).Compared with the EGF group,the expression ofmTORmRNA in EGF combine1,25(OH)2D3group was significantly decreased,thedifference was statistically significant(P＜0.01)；(6)the result of the expression nVDRmRNAshowed:the expression of nVDRmRNA in each group have not seen.(7)the result of theexpression mTOR protein showed:Compared with the normal control group,the expression ofmTOR in EGF group was significantly increased,the difference was statistically significant(P＜0.01),the expression of mTOR in1,25(OH)2D3group was significantly decreased,thedifference was statistically significant(P＜0.01).Compared with the EGF group,the expressionof mTOR in EGF combine1,25(OH)2D3group was significantly decreased,the difference wasstatistically significant(P＜0.01).(8)the result of the expression VDR protein showed:theexpression of VDR in each group have not seen. Conclusion:(1)The1,25(OH)2D3can significantly inhibit the proliferation ofhuman glomerular mesangial cells and promote the apoptosis of them.(2)EGF cansignificantly promote the proliferation of human glomerular mesangial cells and inhibit theapoptosis of them,and the effect of EGF can be inhibited by the1,25(OH)2D3(3)1,25(OH)2D3can significantly inhibit the expression of the mTORmRNAand the mTORProtein in the human glomerular mesangial cells.