The Mechanism Study Of BMP2 In Developmental Dysplasia Of The Hip

Objective: Developmental dysplasia of the hip(DDH)is a common musculoskeletal disease of the newborn,which has a large genetic component.The early diagnosis of DDH is important to prevent the disease.At present,there is no clear gene involved in the pathogenesis of DDH.Previously,we performed genome-wide scanning in 34two-generation families with DDH.Linkage analysis showed that Bone morphogenetic protein-2(BMP-2)was associated with DDH.Bone morphogenetic protein-2 inducible kinase(BMP2K)is a serine/threonine protein kinase.BMP2K is an important regulator of cell differentiation,it is essential for bone development and osteoblast differentiation.Recently,repeated germline mutations and somatic deletions or insertions of BMP2K have been found to be associated with susceptibility to DDH.Two novel mutations in BMP2K may be pathogenic mutations in DDH,a finding that has also been confirmed in sporadic cases of DDH.BMP2K expression was increased during the differentiation of BMP-2-induced prechondrocytes into osteoblast phenotype.It has been found that BMP2K-specific antibody interacts with IRS-2(INSR substrate-2,IRS-2)in co-immunoprecipitation,BMP-2 and IGF-1showed synergistic effects on osteogenic differentiation or new bone formation.Therefore,we hypothesized that BMP-2 and IGF-1 play similar roles in the regulation of cartilage growth and development,and that IRS-2/BMP2K may be an intermediate mediator of RUNX1 regulation by BMP-2,which in turn influences the development of chondrocytes,ultimately affects acetabular development.In order to verify the relationship between BMP-2 and BMP2K in chondrocytes and the regulatory relationship between BMP-2 and BMP2K,the interaction between BMP-2 and BMP2K and its effect on chondrocytes and DDH were tested in vitro and in vivo,it provides a new direction for early diagnosis and treatment of DDH.Methods: 1.To detect the expression of BMP-2 and BMP2K in chondrocytes and the relationship between themThe expression of BMP-2 and BMP2K in rat chondrocytes was detected by qRT-PCR and Western blot,and the interaction between BMP-2 and BMP2K was detected by co-immunoprecipitation.2.After overexpression and knockdown of BMP2,the changes of BMP2K,Irs-2 and ossification index in rat chondrocytes were detectedThe BMP-2 overexpression and knockdown lentiviral vectors were constructed,and the overexpression efficiency of BMP-2 was detected by qRT-PCR.The cell membrane and cytoplasmic protein were separated by using the cell membrane and cytoplasmic protein extraction kit.Empty virus group and BMP-2 overexpression group,empty virus group and BMP-2 knockdown group,empty virus group as control group.The expression of BMP2K was detected by qRT-PCR and Western blot,and the expression of IRS-2 was detected by Western blot,the expression of cartilage ossification-related proteins Col-X,VEGF and ALP were detected by Western blot.3.To detect the expression of BMP-2 and BMP2K in chondrocytes of femoral head in DDH model ratsSixteen 9-week-old Wistar pregnant rats(0 days of pregnancy)were randomly divided into control group and DDH group.Within 12 hours after birth,infant rats were fixed in the position of straight leg swaddling for 4 days,during which the fixed position was removed for 30 minutes every day.All the operations were performed by the same person to eliminate the differences caused by the fixed manipulation of different experimenters.The offspring continued to be breastfed until 2 weeks after birth and were killed by intraperitoneal injection of 20% Chloral hydrate.The intact femoral head and articular cartilage were removed.The expression levels of BMP-2and BMP2K in the articular cartilage of the two-week-old rabbits were detected by Western blot.Conclusion: There was an interaction between BMP-2 and BMP2K in rat chondrocytes.After overexpression of BMP-2,the cartilage ossification was enhanced,the expression of BMP2K was unchanged,and the expression of IRS-2protein was increased,the ability of cartilage ossification decreased,the expression of BMP2K and IRS-2 protein decreased.Both BMP-2 and BMP2K were down-regulated in the DDH model,suggesting that the process of cartilage differentiation to bone was arrested.