| Senile osteoporosis(SOP)is due to imbalance between to age-related osteoclastic bone resorption and osteoblastic bone formation,which has led to bone loss and bone structure changes,and then seriously endangers the health of patients.By the increase of the aging population of society,this problem is particularly prominent.Bone marrow mesenchymal stem cells(BMSCs)decrease osteogenic differentiation with age,and activate osteoclast activity by secreting related factors.Therefore,BMSCs biological characteristics changes play an important role in the pathogenesis of SOP.Previous studies have revealed that the nuclear matrix protein specific AT-rich binding protein 2(SATB2)regulates the age-related changes of BMSCs in human jaw bone.MicroRNA-31(miR-31)can bind to the SATB2 promoter region and silence its expression.Therefore,we propose the following hypothesis:whether miR-31-SATB2 regulates the aging of BMSCs and plays an important role in the development of senile osteoporosis?In this study,we detected the biological characteristics of BMSCs and the expression of miR-31 in BMSCs from different age groups.The effects of miR-31 expression on BMSCs senescence and osteogenic differentiation and activation of osteoclast differentiation were analyzed.Preventive and therapeutic effects of miR-31 inhibitor on aging osteoporosis in rat model were detected.And this will provide a new theoretical and experimental basis for the diagnosis and treatment of SOP.Part ? Analysis of age-related biological characteristics and the expression of miR-31 in BMSCsObjective:To analyze the age-related biological characteristics of BMSCs in different age groups and expression of miR-31 in BMSCs in spontaneous aging of male rats,castration of female rats and replication of cells in vitro.Methods:(1)BMSCs were isolated and cultured from the femoral bone in vitro by the whole bone marrow adherent culture and the culture medium was changed to remove the nonadherent cells.When the monoclonal colony formed,and attached each other's,the cells were passaged.(2)Colony formation assays and Brdu staining for identifing the proliferating function of BMSCs for 8 days.(3)The cell proliferation ability was identified by CCK8 assays.(4)Stemness associated genes expression was detected by Western blot and Immunofluorescence in BMSCs.(5)The multi-differentiation ability was detected by Real-time PCR,Western blot,Oil red stain and alizarin red stain.(6)The cell senescence and associated genes expression was analyzied by Cell Senescence-associated ?-galactosidase Staining and Western blot,and DNA damage by yH2AX fluorescence.(7)Real-time quantitative PCR for miR-31 expression in BMSCs of different age groups,ovariectomized rats and passaged cells.Results:(1)The young group(Y)BMSCs had more clonal colonies than the middle(M)and old group(O),and the BMSCs' proliferation rates gradually decreased by aging.The expression of Nanog,SOX2 and OCT4 for the three master sternness factors was remarkably reduced after aging.(2)BMSCs' osteogenic potential gradually decreased by aging,but the adipogenic potential enhanced.The expression levels of SATB2,Osterix(Osx)and osteocalcin(OCN)in BMSCs were significantly lower than the young and middle-aged groups(P<0.05).The expression of LPL,PPAR-y,C/EBP-a mRNA was higher than Y and M groups(P<0.05).(3)M and O groups observed more SA-?-Gal and y-H2AX positive cells than Y group.Compared to Y group,the senescence-related factors P16,P21 and P53 were revealed markedly higher expression levels.(4)The miR-31 levels in BMSCs were significantly increased after aging,ovariectomy and replicative senescence.Conclusion:The changes of aging characteristics of BMSCs were mainly due to the weakening of stemness and osteogenic differentiation ability,the enhancement of adipogenic differentiation and the increase of cell senescence.In the course of aging of BMSCs,the expression of miR-31 increased,suggesting that miR-31 may be involved in the regulation of BMSCs aging.Part ? Study the effects of miR-31-modified BMSCs in senescence and osteogenesisObjective:To investigate the effect of different levels of miR-31 on the senescence and osteogenic differentiation of BMSCs.Methods:(1)The miR-31 inhibitor was transfected into the old group,and the mimics was transfected into the young group BMSCs.The aging-related phenotypes of BMSCs by ?-galactosidase staining,and DNA damage by the yH2AX fluorescence detection.(2)Western blot for osteogenesis-associated genes such as SATB2,OSX and OCN expression.(3)Alizarin red stain for the osteogenic ability.Results:(1)Reduced SA-?-Gal-positive cells and y-H2AX-positive cells revealed that miR-31 inhibition significantly promoted osteogenic differentiation of older BMSCs,and reduced cellular senescenceas.(2)In contrast,miR-31 overexpression increased cellular senescenceas as shown by induced SA-?-Gal-positive cells and y-H2AX-positive cells,inhibited osteogenic differentiation of BMSCs that osteogenesis associated genes as SATB2,Osx,OCN' expression decreased.Conclusion:MiR-31 is a critical regulator responsible for age-associated osteogenesis of BMSCs.Part ?:BMSCs regulated osteoclast differentiation and function by secreting miR-31Objective:To analyze the expression of miR-31 in the microenvironment of BMSCs and to investigate the effect of miR-31 on the osteoclastic differentiation induced by BMSCs by exosomes.Methods:1)The exosomes were extracted by ultracentrifugation in the cell supernatants.The miRNeasymini kit for the total RNA and Real-time quantitative PCR for the expression level of miR-31.2)DiI as a cell membrane red fluorescent-labeled probe was stained for the BMSCs.The cell supernatants were co-cultured with 4-month-old rat bone marrow.Fluorescence assay was used to verify the mechanism of miR-31 exosomes in BMSCs microenvironment.3)The osteoclast differentiation was induced by co-culture different cell supernatants and 4-month-old rat bone marrow tissue.The growth and function of osteoclasts were assessed by tartrate-resistant acid phosphatase(TRAP)staining.The bone slices were stained for toluidine blue to observe bone resorption by osteoclasts.Results:1)PCR showed a significantly higher expression level of miR-31 in the cell supernatants.2)DiI staining(orange red)showed positive region around nuclear.3)TRAP staining revealed an enhanced osteoclast inductive ability of age-related cell supernatants,and reduced after inhibition of miR-31.4)Bone slices showed greater bone resorption area than Y's medium,also reduced by inhibitor.Conclusions:The expression of miR-31 in the BMSCs microenvironment increased with aging,and the osteoblast differentiation ability in the elderly group was stronger than that in the young group.The inhibition of miR-31 induced osteoblast differentiation and function.The mechanism may be for the aging BMSCs by paracrine encapsulated miR-31 exosomes targeted and regulated osteoclast differentiation and function.Part ? Injection of antagomiR-31 prevented and treated senile osteoporosis in vivoObjective:To investigate the efficacy and feasibility of miR-31 inhibitor in the prevention and treatment of senile osteoporosis in vivo.Methods:We injected antagomiR-31 intraperitoneally into the femur of 15-month-old rats.Micro-CT for detection of bone mineral density and trabecular bone thickness.HE and TRAP staining for detection of bone formation and bone resorption.Results:The BV/TV of the distal femoral bone was significantly higher,the Tb.n increased the Tb.sp decreased after injection.HE staining showed osteogenesis increased,and TRAP revealed osteoclasts decreased significantly.Conclusion:Injection of miR-31 inhibitor can reverse the BMSCs age-related bone resorption and bone loss in vivo,and may become a new means of prevention and treatment of osteoporosis. |
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