The Study Of Klotho On ADSCs Survival And Adipogenic Differentiation

Objective: The aim of this study was to study the effect of secretory Klotho(SKL)on human adipose stem cells(h ADSCs)adipogenic differention and survival.Through isolation and purification of SKL,cell culture,Western Blot,in vivo nude mouse test and other experimental methods,SKL has been proved to promote the survival of h ADSCs,promote adipogenic differentiation,inhibit apoptosis,and regulate the expression of p-AMPK?,LC3 and other proteins,providing a new experimental evidence for clinical repair of soft tissue injury.Methods: 1.293 T cells were cultured in vitro and divided intocontrol group(N group)and Klotho overexpression group.Cells transfected with empty plasmid were used as N group,and cells transfected with plasmid containing SKL gene were used as Klotho overexpression group.Western Blot was used to detect the transfected cell and the secreted Klotho in the culture medium.Then serum free culture medium was collected and Klotho protein was extracted using protein purified nickel column.2.h ADSCs were cultured in vitro,and the cells were evenly spread on the six-well plate.According to the concentration of SKL,the cells were divided into four groups: N group,0.1?g/ml group,1?g/ml group,and 10?g/ml group.The protein expressions of p-AMPK?,AMPK and LC3 in SKL were detected by Western Blot.3.Cells were evenly spread on the sixwell plate,AMPK activators(AICAR)and inhibitors(Compound C)were added according to the groups and divided into seven groups: N group,SKL group,AICAR(A)group,Compound C(C.C)group,SKL+AICAR(SKL+A)group and SKL+Compound C(SKL+C.C)group.cell proteins were extracted after 48 hours.P-AMPK? and AMPK? proteins were detected by Western Blot.4.The cells were divided into four groups: N group,SKL group,A group,and SKL+A group.The cells were cultured in six-well plate and directed differentiation was induced with adipocyte induction solution and stained with oil red O.5.The cells were divided into four groups: N group,SKL group,C.C group and SKL+C.C group.The cells were cultured in six-well plate,and adipogenic induction solution was added to induce directional differentiation of the cells.The oil red O dye solution was dyed to identify the effect of lipid formation.6.The hydrogels were prepared for fat acellular matrix(DAT),nude mouse can be divided into seven groups: DAT group,DAT+h ADSCs group,DAT + h ADSCs + SKL group,DAT + h ADSCs + A group,DAT +h ADSCs + SKL + group A,DAT + h ADSCs + A group,DAT + h ADSCs + SKL + A group,In vivo injection,cells are implanted under the skin,four weeks after Micro-CT inspection,tissue sampling,to histological section,oil red O staining,HE stain and immunofluorescence experiments.Results: 1.Western Blot results showed that SKL protein content in transfected cells and serumfree medium of 293 T cells was significantly higher than that in the control group.The purity and protein content of SKL purified by nickel column reached the required requirements.2.Flow cytometry results showed that SKL promoted the proliferation and survival of h ADSCs and inhibited the apoptosis of h ADSCs.3.Western Blot results showed that with the increase of SKL concentration,the protein expression of p-AMPK? in h ADSCs decreased gradually,while the protein expression of LC3 increased gradually.During adipogenesis of h ADSCs,the expression of secretory Klotho protein increased gradually.4.Oil red O staining of cell slides confirmed the presence of a large number of lipid droplets in SKL group,and SKL promoted h ADSCs adipogenic differentiation.5.In vivo experiment,h ADSCs treated with different drugs were fused with DAT and implanted into the body.Micro-CT detection was performed 4 weeks later,samples were collected and fixed,freezing microtome section were made,and oil red O staining confirmed that the SKL group was rich in lipid droplets and had significant lipid formation effect.The AICAR group inhibited adipogenesis,while the Compound C group inhibited adipogenesis,which further confirmed that the effect of SKL on adipogenesis was not completely through the AMPK pathway.Immunofluorescence results showed that SKL promoted the upregulation of C/EBP?,and SKL group had the strongest fluorescence effect.Conclusions: The results showed that SKL significantly promoted the survival and autophagy of h ADSCs,and SKL may promoted the adipogenic differentiation of h ADSCs by down-regulating the expression of p-AMPK? protein.