Effect Of Active Vitamin D₃ On The Expression Of MTOR/p70s6K In Human Glomerular Mesangial

Objective:The aim of this study was to investigate the expression of m TOR/p70s6 k signaling pathway in human mesangial cells; To investigate the effects of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on cell proliferation in human glomerular mesangial cells and it’s effects in the regulation of glomerular mesangial cells of the m TOR/p70s6 K signaling pathways;To provide a solid theoretical foundation for using vitamin D3 to treat the chronic kidney disease in clinical.Methods:The cultured human mesangial cells, which was subcultured 3~7 generations,were divided into four groups:normal control group,VD3group(plus 10-8mol/L of 1,25-dihydroxyvitamin D3),Rapamycin group(plus 5μg/m L of rapamycin) and VD3+ Rapamycin group(plus 10-8mol/L of 1,25-dihydroxyvitamin D3 and 5μg/m L of rapamycin),treatment of 48 h. The morphology and growth of human mesangial cells were observed by inverted phase contrast microscope; m TOR and p70s6 K expression was detected by immunofluorescence, m TOR m RNA and P70S6 K m RNA expression was detected by real-time fluorescence polymerase chain.Result:(1) Cell morphology and growth in each group. Normal control group mesangial cell body is spindle shaped, irregular star or dendritic, nucleus in the middle, round or oval in shape, with clear; 1,25(OH)2D3 group mesangial cells, a small number of mesangial cell shrinkage smaller, nuclear enrichment punctate, condensed cytoplasm and vacuoles, part of the cells floating; Rapamycin and rapamycin combin 1,25(OH)2D3 group mesangial cells, the number of nuclei was significantly reduced, and the length of spindle was prominent or disappeared.(2)The expression of m TOR and p70s6 K in glomerular mesangial cells was detected by immunofluorescence assay after 48 h intervention.Compared with normal control group, the expression of m TOR and p70s6 K in 1,25(OH)2D3 group, rapamycin group and rapamycin combin 1,25(OH)2D3 group were significantly recudced, the difference was statistically significant(p<0.01);compared with 1,25(OH)2D3 group, the expression of m TOR and p70s6 K in rapamycin group had no statistically significant(p>0.05), compared with 1,25(OH)2D3 and rapamycin group, rapamycin combin 1,25(OH)2D3 group were significantly recudced, the difference was statistically significant(p<0.01).(3)The expression of m TOR m RNA and p70s6 K m RNA in glomerular mesangial cells was detected by real-time fluorescence polymerase chain. Compared with the normal control group, the expression of m TOR m RNA in 1,25(OH)2D3 group, rapamycin group and rapamycin combin 1,25(OH)2D3 group were significantly recudced, but p70s6 k m RNA were significantly increased,the difference was statistically significant(p<0.05);compared with 1,25(OH)2D3 and rapamycin group, the expression of m TOR m RNA in rapamycin combin 1,25(OH)2D3 group were significantly recudced, but p70s6 k m RNA were significantly increased,the difference was statistically significant(p<0.05).Conclusions:(1)1,25(OH)2D3 can inhibit the proliferation of human glomerular mesangial cells significantly;(2)m TOR/p70s6 K signaling pathway exist in human mesangial cells, 1,25(OH)2D3 inhibit mesangial cells proliferation by regulating the pathway;(3)1,25(OH)2D3 can cooperate with rapamycin to inhibit m TOR/p70s6 K signaling pathway, strengthen to inhibit glomerular mesangial cells proliferation;(4)1,25(OH)2D3 probably affect protein expression by regulating association protein translation process in mesangial cells, thereby inhibiting the proliferation of glomerular mesangial cells, inducing cells apoptosis.