The Effects Of BMSCs On The MTOR Of Adriamycin-induced Podocytes

Objective: To investigate the effects of BMSCs on the m TOR of adriamycin-induced podocytes.Method:BMSCs were extracted form SD rat bone marrow by the method of density gradient centrifugation combined with adherence separation.Trough the passage and the amplification to P3 generation and then the antigen markers including CD29,CD34,CD45 and CD90 on the BMSCs surfaces were tested by flow cytometry method.The function of BMSCs were identified by chondrogenic inducing reagents and adipogenic inducing reagents.The rat primary podocytes were extracted form the kidney of SD rats by graded sieving method combined with IV enzyme digesting method and cultured in vitro.Nephrin and podocin epitope of cells were detected by immunofluorescence.In vitro experiments, divided into three. normal group:podocytes; adriamycin group: podocytes + adriamycin(2ug/ml); BMSCs group: podocytes + BMSCs(cultured 24h) + adriamycin(2ug/ml).; After all experimental groups intervention 0h, 3h, 24 h,Western bolt detected the expression of nephrin which was podocyte cytoskeleton-associated protein and the experssion of m TOR, p- m TOR, raptor, rictor which were the associated protein of the m TOR signal pathway.Result:BMSCs are spindle,polygonal fibroblast-like morphology and the same size under the microscope.BMSCs are successfully induced into cartilage cells and fat cells through chondrogenic inducing reagents and adipogenic inducing reagents.Using the flow cytometry method to test BMSCs,the surface markers CD29 and CD90 are positive.otherwise,the surface markers CD34 and CD45 are negative.The purity of BMSCs is 98%.The glomerulus can be obtained by graded sieving method.The podocytes grow from the glomerular digested by IV collagenase,which are cobblestone-like cells.Immunofluorescence method is used to detect the surface.Nephrin and podocin canbe expressed.Western bolt analysis showed that:at 0 hours and 3 hours, the normal group, the adriamycin group, the BMSCs group have no difference between the nephrin and rictor’s expression;At 24 hours, the expression of nephrin in the BMSCs group and the adriamycin group were less than the normal group, but the expression of the BMSCs group were more than the adriamycin group.At the same time,the expression of rictor in the BMSCs group and the adriamycin group were more than the normal group,but the expression of the BMSCs group were less than the adriamycin group;m TOR, p- m TOR, raptor protein at 0 hours, the normal group,the adriamycin group and the BMSCs group have no obvious difference, but at 3 hours, 24 hours, the expression of the BMSCs group and the adriamycin group were more than the normal group, but the expression of the BMSCs group were less than the adriamycin group, the expression quantity of 24 hours are all more than 3 hours.Conclusion:By using density gradient centrifugation and adherent cells in vitro cultivation method can successfully extract the BMSCs.They are high-purity, high-activity,high-proliferative,pluripotent and meet the requirement of experiment.The available podocytes for lab can be cultured trough graded sieving method combined with IV enzyme digestiong method.The mechanism of AMD damage podocyte is associated with the excessive activation of m TOR, resulting in podocyte function disorder.Rat BMSCs could inhibit m TOR activity to protect adriamycin damage cells and increase the nephrin protein expression.