| Background:Currently,adipose-derived mesenchymal stem cells(ADSCs),due to their large distribution in the human body,rapid osteogenic differentiation in vitro,and low immunogenicity,It is considered to be one of the most promising stem cells for Osteoporosis(OP).However,the differentiation ability of ADSCs after in vivo transplantation is insufficient.Currently,the research field mainly focuses on the regulation of osteogenic differentiation of stem cells after in vivo transplantation,and no clinical trials have been conducted yet.In view of the etiology and pathogenesis of kidney deficiency and pulp deficiency in osteoporosis,scholars of traditional Chinese medicine put forward that acupuncture and moxibustion with the function of tonifying kidney and pulp can prevent and treat osteoporosis.Meanwhile,osteogenic differentiation of ADSCs was related to the acetylation level of histone H3K9.Acupuncture and moxibustion is widely used in the prevention and treatment of osteoporosis because of its function of regulating stem cell differentiation and promoting histone acetylation.However,acupuncture regulation on osteogenic differentiation of ADSCs has not been reported.Therefore,it is the main content of this study to explore whether the internal mechanism of electroacupuncture combined with autologous ADSCs system transplantation promoting bone regeneration and increasing bone code is related to the acetylation level of histone H3K9Objective:To explore the regulation of acetylation of histone H3K9 by electroacupuncture after transplantation of ADSCs system,activate the expression of osteogenic genes,improve the overall effect of osteoporosis,and clarify the possible mechanism of electroacupuncture promoting osteogenic differentiation of ADSCs in vivo and improving osteoporosis.Methods:ADSCs were isolated from the groin of rats and cultured to obtain P3-ADSCs.The surface antigen was detected by flow cytometry,and the bone differentiation was observed by alizarin red staining,and the lipid differentiation was observed by oil red O staining.After the transfection of lentivirus Zs-green-ADSCs into P3-ADSCs,the stable transfection strains of Zs Green-ADSCs were screened by purinase.Forty-two female SD rats were purchased,and 6of them were randomly selected and assigned to the Sham group.The ovaria-like adipose tissue was resectomized,and the other rats were resectomized bilateral ovarian tissue to establish osteoporosis model rats.After 8 weeks,the model was established,and then the rats were randomly divided into 6 groups with 6 rats in each group.Model group(M)was injected with1 ml normal saline.Electroacupuncture group(EA)was given electroacupuncture therapy,3times a week;The ADSCs transplantation group(ADSCs)was injected with 1ml autologous Zs Green-ADSCs;In the electroacupuncture +ADSCs transplantation group(EA +ADSCs),1ml autologous Zs Green-ADSCs were injected at the same frequency as the electroacupuncture group.Deacetylase inhibitor group +ADSCs(TSA +ADSCs)was intraperitoneally injected with TSA and 1ml autologous Zs Green-ADSCs;Deacetylase agonist group +electroacupuncture +ADSCs transplantation group +(ITSA+ EA +ADSCs),ITSA intrabitoneal injection,electroacupuncture treatment,at the same frequency as the electroacupuncture group,at the same time injection of 1ml autologous Zs Green-ADSCs,8 weeks after intervention,in vivo imaging to observe the enrichment of green fluorescence signal in the femoral head region of rats in each group.Micro-CT,HE staining and immunohistochemical ALP staining were used to observe the changes of femur density,microstructure and tissue morphology in each group.Serum estradiol(E2)and alkaline phosphatase(ALP)levels were determined by ELISA.Expression levels of BMP-2,OSX and Runx2 genes in each group by RT-q PCR;The protein expression levels of H3,Ac-H3K9,Runx2,BMP-2 and OSX were detected by Western-blotting.Results:1.P3-ADSCs were identified successfully,green fluorescence signal could be seen after lentivirus transduction,and the system could be enriched in rat femur after transplantation.2.Compared with the Sham group,group M rat femur bone mineral density to reduce that successful osteoporosis rats model is set up.3.Compared with group M,ADSCs group rats femur bone density increases,osteoblast number increase,decrease serum ALP,E2,osteogenesis related gene and protein BMP-2,Osteri X,Runx2 expression level was significantly elevated(P<0.01),indicating that ADSCs transplantation can improve osteoporosis in castrated rats.4.Compared with ADSCs group,the TSA + ADSCs group rats femur bone density increases,osteoblast number increase,decrease serum ALP,E2,osteogenesis related gene and protein expression was significantly elevated(P<0.01),the expression level of Ac-H3K9 was significantly increased(P< 0.01),suggesting that activation of Ac-H3K9 could up-regulate the expression of osteogenic genes and improve osteoporosis.5.Compared with the M group,EA group rats femur bone density increases,osteoblast number increase,decrease serum ALP,E2,osteogenesis related gene and protein expression was significantly elevated(P<0.01),there was no significant difference in H3 expression(P>0.05),the expression level of Ac-H3K9 was significantly increased(P<0.01),indicating that electroacupuncture can activate Ac-H3K9 to up-regulate the expression of osteogenic gene and improve osteoporosis.6.Compared with ADSCs group,EA + ADSCs group rats femur bone mineral density increased,osteoblast number increase,decrease serum ALP,E2,a significant rise in osteogenesis related genes and proteins(P<0.01),the protein expression of Ac-H3K9 was significantly increased(P< 0.01),indicating that electroacupuncture combined with autologous transplantation to activate Ac-H3K9 can up-regulate the expression of osteogenic genes and proteins,and improve osteoporosis.Compared with EA + ADSCs group,ITSA + EA + ADSCs group rats femur bone mineral density is reduced,the decrease in the number of osteoblast,elevated serum ALP,E2,reduced osteogenesis related gene and protein expression,Ac-H3K9 protein expression decreased,prompt acusector joint activation ADSCs transplantation Ac-H3K9 expression,The effect of upregulation of osteogenic gene expression can be partially inhibited by ITSA.Conclusion:Electroacupuncture can further enhance the osteogenic differentiation ability of ADSCs in vivo and improve the osteoporosis status of rats.The underlying mechanism may be related to electroacupuncture specifically regulating the acetylation level of histone H3K9 and upregulating the expression levels of osteogenic genes and proteins. |
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