| Experimental purpose:Bone marrow mesenchymal stem cells(BMSCs)are pluripotent stem cells with osteogenic and adipogenic differentiation ability,which is regulated by a variety of factors.Previous studies have found that hyperlipidemia can lead to reduced bone formation and poor osseointegration around implants in rats.Vacuolar sorting proteins26(VPS26)promotes the growth of Arabidopsis thaliana and is responsible for material transport in biological vesicles.However,the function of VPS26 in cell proliferation and differentiation is still unknown.Studies have shown that VPS26 can transport Wnt protein,which is crucial in promoting BMSCs differentiation.The objectives of this study were as follows:① to explore the effect of VPS26 on BMSCs osteogenesis and adipogenesis.②to confirm that VPS26 regulates the metabolism of BMSCs through the Wnt/β-catenin pathway.③to study the effects of VPS26 on ectopic osteogenesis and osseointegration around implants in hyperlipidemia rats.Experimental methods:(1)To study the effect of VPS26 on BMSCs differentiation:① BMSCs were induced under high fat/normal environment,and the expression of VPS26 was detected by RT-PCR.② VPS26 was overexpressed in BMSCs,and the expression of osteogenic differentiation indexes ALP and RUNX2,lipid differentiation indexes PPAR-y and FABP4,β-catenin and CCND1 in Wnt pathway were detected by RT-PCR and Western blot.ALP and oil red O staining were used to observe the effect of VPS26 on osteogenic and adipogenic differentiation of BMSCs.③ VPS26 was overexpressed in BMSCs and then silenced,and the expression of osteogenic and adipogenic differentiation indexes and Wnt-related factors were determined by rescue experiment.(2)To study the specific mechanism of VPS26 affecting BMSCs differentiation:①the expression of VPS26 and β-catenin was detected by COIP after the protein samples of BMSCs were extracted.② the expression of VPS26 and β-catenin was detected by immunofluorescence co-location.③ After BMSCs had low/overexpression of VPS26,Top/FOP was transfected and Wnt3a was added.The expression of Wnt-related factors was determined by TOPFLASH luciferase gene reporting and Western blot analysis.(3)To study the effect of VPS26 on osteogenesis and osseointegration around implants in hyperlipidemia rats:① the femur of hyperlipidemia rats and normal rats were detected the expression of VPS26 in cells was detected by IHC.② VPS26 was overexpressed/knockdown in BMSCs and loaded with HA/TCP scaffold.After 7 days of osteogenic induction,VPS26 was implanted subcutaneously in nude mice.Hard tissue sections were stained with HE to detect the effect of VPS26 on bone metabolism,and the expression of β-catenin was observed by IHC.③ the hyperlipidemia model was successfully constructed.After VPS26 was overexpressed in hyperlipidemia rats,the expression of osteogenic and adipogenic differentiation indexes were detected by RT-PCR.Bone formation and adipogenesis around implants were detected by micro-CT and hard tissue section staining.Experiment results:(1)High fat environment inhibited the expression of VPS26:After high fat osteogenesis induced BMSCs,the expression of VPS26 in BMSCs was continuously decreased,and the expression of VPS26 in normal osteogenesis induced BMSCs was continuously increased.(2)Overexpression of VPS26 regulates BMSCs osteogenic and adipogenic differentiation:After VPS26 was overexpressed in BMSCs,the expression of VPS26,β-catenin,CCND1 and Wntless were increased in LV-VPS26 group,compared with LV17 group,the expression of osteogenic indexes ALP and RUNX2 were increased,the expression of adipogenic differentiation indexes FABP4 and PPAR-y was decreased,and the number of mineralized nodals was increased.The number of lipid droplet formation decreased,and the osteogenic differentiation ability of BMSCs increased,but the adipogenic differentiation ability decreased.(3)Inhibited the expression of VPS26 in BMSCs after overexpressing can reverse its effect on differentiation:After overexpression of VPS26 in BMSCs,the expression of VPS26 was inhibited again,and the expression of β-catenin and CCND1 was decreased,the expression of RUNX2 was decreased,and the expression of FABP4 and PPAR-y were increased.(4)VPS26 plays a role by binding β-catenin:① Immunoprecipitation showed that VPS26 and β-catenin co-located in BMSCs.② Immunofluorescence co-localization showed that VPS26 was co-located with β-catenin in BMSCs.③TOPFLASH showed that the TOP/FOP value of the LV-VPS26 group was higher than that of the LV-nc group after adding Wnt3a.Compared with the shscr group,the TOP/FOP value of the shVPS26 group was decreased.④ After the low expression of VPS26 in BMSCs,the expression of β-catenin in the shVPS26 group did not change significantly compared with that in the shscr group after the addition of Wnt3a.(5)Overexpression/underexpression of VPS26 affected subcutaneous ectopic bone formation in nude mice:① Compared with the LV-nc group,new bone formation was significantly increased in the LV-VPS26 group.② Compared with the shscr group,the formation of new bone in the shVPS26 group was significantly reduced.(6)Overexpression of VPS26 inhibited bone malassociation caused by hyperlipidemia in rats:Immunohistochemical results of rat femur showed that the expression of VPS26 in osteoblasts of high-fat rats was significantly lower than that of normal rats.Compared with the LV-nc group,the formation of new bone around implants in the LV-VPS26 group increased,and fat formation decreased.Moreover,the expression of osteogenic differentiation index ALP increased,and the expression of adipogenic differentiation index PPAR-y and FABP4 decreased.Conclusion:1.Overexpression of VPS26 promote osteogenic differentiation and inhibited adipogenic differentiation of BMSCs.2.VPS26 can promote ectopic osteogenesis and osseointegration around implants in hyperlipidemia rats.3.VPS26 regulates the osteogenic and adipogenic differentiation of BMSCs through Wnt/β-catenin pathway. |
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