| Along with the development of the society,there is more and more ageing population.It should be concerned for the osteoporosis and osteoporotic fractures due to ageing.Osteoporosis is a polygenic disease.The effects among the multiple genes are very important to bone metabolism.Of note,as age advances less new bone is formed than resorbed in each site remodelled,producing bone loss and structural damage.Bone anabolic synthesis is mainly come from the osteogenesis of stem cells.Therefore,the osteoporosis and fracture healing are much associated with the age-related genes.To understand the pathogenesis of osteoporosis and to deal with the osteoporotic fractures,it is very important to figure out the regulating mechanism of various genes to the osteogenic differentiation of stem cells.Interestingly,previous studies have demonstrated that IGFBP7 was closely related with stem cells senescence and bone metabolism.However,the effects of IGFBP7 to the osteogenic differentiation of stem cells remains unclear.Thus,in this study,we investigated the differential expression of IGFBP7 in bone marrow-derived mesenchymal stem cells(BMSCs)between young group and older group.Meanwhile,in vitro,the influence of IGFBP7 to the osteogenesis of BMSCs were studied and its possible mechanism were also investigated.In addition,using a rat tibial defect model,it were confirmed that a sheet of IGFBP7-overexpressing BMSCs improved bone defect healing in vivo.This study has been divided into three parts:(1)The differential expression of IGFBP7 in BMSCs between young people and the aged;(2)The effects of IGFBP7 to the osteogenic differentiation of BMSCs;(3)The effects of IGFBP7-overexpressing BMSCs sheet to the tibial defect.Chapter I The differential expression of IGFBP7 in human BMSCs between young people and the agedObjective:This study aimed to investigate the differential expression of IGFBP7 in human BMSCs between young people and the aged.Methods:BMSCs were isolated and purified by density gradient centrifugation.Characteristic cell surface markers were identified by flow cytometry.The isolated cells were induced with adipogenic induction medium,osteogenic induction medium,and chondrogenic induction medium to confirm their sternness.The people were divided into two groups,namely young group aged 16 to 22;the older group with more or equal to 70 years old.The expression of IGFBP7 in serum between the older group and the young group was detected by ELISA.The differential expressions of osteogenic specific genes,IGFBP7,and β-catenin between the older group and the young group by RT-qPCR and Western blot analysis.Meanwhile,the comparison of different passages of BMSCs(P1,P7,P15)in osteogenic specific genes,IGFBP7,and β-catenin.Results:The positive rates of CD73,CD105,CD109,CD34,CD45,and CD31 of isolated cells were 99.8%,99.9%,98.5%,0.042%,0.06%,and 0.051%,respectively.Lipid droplets,mineral desposits,and extracellular matrix of cartilage were detected successfully after induction in vitro.Higher concentration of IGFBP7 were found in serum in older group by ELISA analysis(P<0.05).The expression of osteogenic specific genes(RUNX2,OCN,SP7)and β-catenin were lower in older group than thosein the young group(P<0.05),however,higher expression of IGFBP7 was detected in old group(P<0.05).Following the increasing passages,the expression of osteogenic specific genes and(3-catenin were also decreased,nevertheless,more IGFBP7 was oberved(P<0.05).Conclusion:Therefore,the increased expression of IGFBP7 and the decreased osteogenic specific genes were found with age.The Writ/β-caterin signaling pathway were alsoreduced with age.Chapter II The effects and mechanism of IGFBP7 in the osteogenic differentiation of BMSCsObjective:The objective of this study is to investigate the effects and related mechanism of IGFBP7 in the osteogenic differentiation of BMSCs.Methods:IGFBP7 were overexpressed and down-regulated by lentivirus vector.These BMSCs were divided into four groups,IGFBP7-overexpressing group(exlg7),LV5-NC group,IGFBP7-downregulating group(shlg7),and LV3-NC group.The effects of this gene in proliferation rate of BMSCs by trypan blue staining.The effects of IGFBP7 in the expression of osteogenic specific genes among these groups were detected by RT-qPCR,Western blot analysis,and immunofluorescence(IF).ALP activity were observed by ALP activity detecting kit.The differential results among those groups in mineral deposits were also detected by alizarin red staining and von Kossa staining.Meanwhile,related signalling pathways were examined.In addition,inhibitory effects were detected to confirm the signalling pathway.Results:Compared with LV3-NC group,the lower proliferation rate was in shlg7 group compared with the other groups(P<0.05),and no significant difference was found between exIg7 group and LV5-NC group(P>0.05).Compared with control group,higher expression of osteogenic specific genes were found in the exIg7 group(P<0.05),and lower expression of osteogenic specific genes were found in the shlg7 group(P<0.05)at days 3 and 7 of osteogenic differentiation.Meanwhile,at day 1 of osteogenic differentiation,similar results were also found.ALP activity was increased due to the overexpression of IGFBP7.More mineral desposits were observed in exIg7 group and less mineral deposits were found in the shIg7 group.The enhancing effect of extracelluar IGFBP7 protein to the osteogenesis of BMSCs was concentration dependent manner.The specific osteogenic genes and mineral deposits were promoted with more than 500 ng/mL extracellur IGFBP7.No significant difference were detected among each group MAPK(ERK/p-38/JNK)signaling pathway and PI3K/Akt signal pathway.Compared with control group,more total β-catenin and actived β-catenin were found in exIg7 group,and less total β-catenin and actived β-catenin were found in shIg7 group.In addition,the increased of osteogenic differentiation due to the overexpression of IGFBP7 were partly decreased by the specific Wnt/β-catenin signalling inhibitors(DKK1 and siRNA).Conclusion:IGFBP7 regulated the osteogenic differentiation of BMSCs via Wnt/β-catenin signalling pathway.Chapter III The effect of a sheet of IGFBP7-overexpressing BMSCs to the rat tibial defect healingObjective:To explore whether a sheet of IGFBP7-overexpressing BMSCs could enhance the rat tibial defect healing.Methods:The BMSCs sheet were constructed in vitro.A total of 30 SD rats were divided into 3 groups:blank group,ctrl group,and the IGFBP7-overexpressing group,and 10 rats each group.BMSCs sheets were transplanted into the rat tibial defect model.After postoperative 8 weeks,euthanasia were performed in these animals.The healing of tibial defect were observed by X ray and Mirco-CT.Histological analysis were also performed to evaluate the healing of bone defect,including HE staining,safranin O and fast green staining,Masson staining,and immunohistochemical analysis.The transplant cells were traced by GFP immunohistochemical analysis.Results:X ray taken at 8 weeks showed that the cortical gap was clearly present in the blank group.In the control group,this gap was obscure,and more bridging callus formation was evident at the fracture site.compared with the blank group.In the BMSCs overexpressing IGFBP7,the gap had disappeared.The results of micro-CT indicated significantly more bone formation in the BMSCs overexpressing IGFBP7 than in control group;moreover,among the three groups,the gap was widest in the blank group.Quantitatively,the model treated with a sheet of BMSCs overexpressing IGFBP7 displayed a significant increase in the BV/TV compared with those of the other two groups.Histological analysis showed that the gaps were filled with fibrous tissue and a few chondrocytes;moreover,no bridging bone formation at the nonunion site was observed in the blank group.In the control group,a thick callus consisting of newly formed woven bone tissue was detected at the nonunion site,and the callus was undergoing remodeling.In the BMSCs overexpressing IGFBP7,the defect sites were sealed,and the remodeling of the callus was almost complete,indicating bony healing of the fracture.Immunofluorescence analysis showed that significantly more COL1A1 and OPN appeared at the fracture site in the BMSCs overexpressing IGFBP7 than in the other two groups.Sporadic of GFP expression was found in the control group and in the BMSCs overexpressing IGFBP7.Conclusion:A sheet of IGFBP7-overexpressing BMSCs promoted the healing of bone defect. |
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