Study On Bmscs Mediated The Repair Of Damaged Podocytes By EGFR Pathway

Objective: To explore the signal pathway of BMSCs regulating the repair of podocyte cytoskeleton protein.Methods: 1.BMSCs were extracted from the femur of SD rats by the adherent culture of whole bone marrow.They can be used for experiments when they were cultured to the third generation.The function of BMSCs were identified by osteogenesis and adipogenesis inducing reagents in vitro.2.Podocytes were extracted from the kidneys of SD rats by graded sieving method combined with IV enzyme digesting method,and their specific proteins Synaptopodin and Podocin were detected by immunofluorescence.3.The content of EGF in the supernatant of BMSCs was detected by ELISA.4.Adriamycin hydrochloride with concentration gradient of 0.5μg/ml,1μg/ml,2μg/ml and 4μg/ml was set as the control group.In addition,the experimental group with the same concentration of adriamycin+10ng/ml EGF was set.CCK-8 was used to detect the activity of podocytes at different time points within 48 h.5.The differentiated podocytes were divided into four groups: normal group: podocyte;adriamycin group: podocyte + adriamycin(2μg/ml);stem cell group: podocyte + adriamycin(2μg/ml)+ BMSCs(co-cultured for 24 hours);epidermal growth factor(EGF)group: podocyte + adriamycin(2μg/ml)+ EGF(10 ng/ml).The podocytes were collected in each experimental group after intervention for 24 hours.The expression of EGFR,p-EGFR,AKT,p-AKT,m TOR,p-m TOR and synaptopodin were detected by Western blot.Results:1.BMSCs are spindle,polygonal fibroblast-like morphology and the same size under the microscope.BMSCs are successfully induced into osteocytes and adipocytes through osteogenic inducing reagents and adipogenic inducing reagents.2.The glomerulus can be obtained by graded sieving method.After digestion with IV collagenase,the glomeruli were cultured for 3 days,which were cobblestone-like cells with large cell body and irregular foot like protuberance on the surface.Immunofluorescence staining showed that podocytes could combine with specific antibodies of synaptopodin and podocin,and displayed corresponding fluorescence under excitation light.3.When BMSCs were cultured in vitro,EGF could be released to the surrounding environment,and the increase of the concentration of the latter was fast at first and then slow with time,and it was stable at 12000-13000 pg / ml around 24-48 h.4.When 2 μg/ml adriamycin was applied to podocytes for 24 hours,adriamycin inhibited the proliferation of podocytes most obviously,and the effect of 10 ng / ml EGF on the repair of podocytes was also the most significant.5.The results of Western blot showed that after 24 hours of intervention,the levels of p-EGFR,p-Akt,m TOR,p-m TOR and synaptopodin in adriamycin group were significantly lower than those in normal group.The gray level of each protein in the stem cell group also decreased in different degrees,but the decrease was small compared with the adriamycin group;the gray level of each protein in the EGF group also decreased compared with the normal group,and the change degree was similar to that in the stem cell group.Conclusion:1.BMSCs can be successfully isolated and cultured from rat femur by adherent culture of whole bone marrow,which are high-purity,high-activity,high-prolifer ative,pluripotent and meet the requirement of experiment.2.Podocytes can be extracted from rat kidney by differential screening and IV collagenase digestion,and the specific protein synaptopodin and podocin can be expressed in vitro.3.Co-culture of BMSCs with podocytes in vitro can repair the damage of podocyte cytoskeleton protein Synaptopodin induced by Adriamycin.BMSCs can up-regulate the phosphorylation level of EGFR on the surface of cell membrane,and cause a series of downstream related proteins including p-AKT,m TOR and p-m TOR to change to the same extent,thus promoting the transcription of cell proteins and the formation of cytoskeleton proteins.The effect is similar to that of EGF alone.This phenomenon reveals that BMSCs can play an anti-injury role by secreting EGF to activate EGFR pathway in podocytes and promote protein transcription and translation.