| BackgroundOsteoarthritis(Osteoarthritis,OA)is a common degenerative joint disease,with cartilage extracellular matrix components by excessive degradation,which leads to cartilage destruction as the main feature,and accompanied by joint marginal osteophyte formation.Because chondrocytes belong to terminally differentiated cells,their repair ability is limited,and it is difficult to regenerate after destruction,there is still lack of satisfactory treatment.Bone marrow mesenchymal stem cells(BMSCs)are multipotent adult stem cells,which have the ability to differentiate into cartilage,bone,fat,tendon and ligament.And BMSCs have many advantages,including abundant source,convenient material collection,rapid proliferation and strong differentiation ability,which have become one of the best seed cells for cartilage tissue repair.In addition,neural cell adhesion molecule(NCAM)in bone marrow mesenchymal stem cells has been found,and may be involved in the regulation of the differentiation of mesenchymal stem cells.Though NCAM is highly expressed in the process of BMSCs differentiate into chondrocytes,its specific role and mechanism are not fully understood yet.The aim of this study is to clarify the role and mechanism of NCAM in the differentiation of BMSCs into chondrocytes.Objective1 To study the effect of neural cell adhesion molecule(NCAM)on the differentiation of bone marrow mesenchymal stem cells(BMSCs)into chondrocytes;2 To make sure the effect of NCAM on differentiation of cartilage via the differentiation from chondrocytes to chondrocyte precursor cells(ATDC5).3 To investigate the signaling pathways of NCAM on the differentiation of bone marrow mesenchymal stem cells(BMSCs)into chondrocytes;4 To study the relationship between NCAM and osteoarthritis.Methods1 Cells culture and differentiation,the establishment of NCAM knockout BMSCs(KO cells)were studied in vitro.2 The changes of RunX2 and Col? in chondrocyte hypertrophy were determined by Western blot,qPCR and alizarin red staining after adding specific chondrocytes in normal(WT)and KO BMSCs for 0d,1d,3d,7d,10 d and 14 d,and analyze whether the expression levels of RunX2 and Col? have changed.3 The NCAM interference plasmids were transfected into ATDC5 cells.Western blot and alizarin red staining were used to detect the changes of chondrocyte hypertrophy markers,such as RunX2 and COL?.It was confirmed that NCAM was involved in the differentiation of chondrocytes.4 In WT cells transfected with EMEK and U0126,a specific inhibitor of ERK kinase was added to KO cells s,for determining the changes of chondrocyte hypertrophy markers RunX2 and COL?,and confirming that ERK signaling pathway is involved in chondrocyte differentiation as well by Western blot,qPCR and alizarin red staining.5 The rat OA model was induced by mono-iodoacetate(MIA).The tissue protein of articular cartilage was extracted and the changes of NCAM were detected by Western blot to analysis of NCAM expression in osteoarthritis.6 IL-1? was added to ATDC5 cells to establish a cell inflammatory model,western blot was used to detect the changes of NCAM in the differentiation of BMSCs into chondrocytes under inflammatory conditions in vitro.7 NCAM was overexpressed in IL-1?-induced ATDC5 cells,western blot and immunofluorescence were used to detect the changes of RunX2 and Col? in chondrocyte hypertrophy.Results1 Previous studies have shown that the expression of NCAM in normal BMSCs is significantly increased with the increase days of chondrocyte differentiation.Therefore,we established BMSCs of knockouting NCAM for studies in vitro.The results showed that,the expression of chondrocyte hypertrophy markers,RunX2 and COL? were significantly up-regulated,and the color of alizarin red staining was deepened when NCAM was defected.These results suggested that deletion of NCAM could promote hypertrophy chondrocyte differentiation of BMSCs.2 In order to further confirm that deficiency of NCAM can promote hypertrophy chondrocyte differentiation,we silenced NCAM in ATDC5 cells that are chondrocytes differentiate precursor cells.The result showed that interference of NCAM could obviously promote the differentiation of hypertrophy chondrocytes.3 In order to explore the molecular mechanism of deficiency of NCAM promotes hypertrophy chondrocyte differentiation,we made a research about the signaling pathways.The results showed that the expression of p-ERK was significantly up-regulated after deletion of NCAM;we found chondrocyte hypertrophy markers RunX2 and COL? were obviously down-regulated or up-regulated when ERK signaling pathway inhibition or activation respectively.These results suggested that deletion of NCAM may promote the differentiation of hypertrophic chondrocytes through ERK signaling pathway.4 We found in OA model that the expression of IL-1 beta and TNF-alpha was up-regulated,the expression of NCAM was significantly reduced in rat OA model,which was induced by sodium iodoacetate.In addition,IL-1 beta was added into ATDC5 cells to simulate the environmental conditions of osteoarthritis in vitro,the results showed that the expression of NCAM was significantly decreased after the addition of IL-1 beta,whereas the expression of RunX2,a marker of chondrocyte hypertrophy,was significantly increased.These results indicate that the expression of NCAM is down-regulated in the model of OA in vivo and in vitro.5 In order to further explain whether overexpression of NCAM could relieve the symptoms of osteoarthritis,we overexpressed NCAM in IL-1 beta induced ATDC5 cells.The results showed that the overexpression of NCAM,chondrocyte hypertrophy markers RunX2 and COL? obviously reduced.These results suggested that NCAM plays an important role in osteoarthritis.Conclusion1 Deficiency of NCAM promotes the differentiation of hypertrophic chondrocytes.2 ERK pathway is involved in NCAM regulation of hypertrophic chondrocyte differentiation.3 NCAM regulates inflammation-induced hypertrophic chondrocyte differentiation,suggesting that NCAM plays an important role in osteoarthritis. |
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