Study On The Effects Of Ihh Signal Blocking And Cell Co-culture On Growth Of Condylar Chondrocytes And The Related Mechanism

Objective The purpose of the present study is to establish the culture model of rabbits’ condylar chondrocytes;to study the effects of Ihh signal blocking on the growth of rabbits’ condylar chondrocytes and its related mechanism;to study the effects of co-culture of bone marrow mesenchymal stem cells(BMSCs)with chondrocytes on BMSCs.Method 1)Condylar chondrocytes were cultured from 4W healthy New Zealand white rabbit,using Alcian blue staining and Toluidine blue staining to identify the chondrocytes.2)Condylar chondrocytes were treated by the different concentration of cyclopamine to blocking the Ihh signal.CCK-8 was used to evaluate the proliferation of the cells;the effects on the cell cycle、apoptosis and differentiation was detected by flow cytometry.3)Rat bone marrow mesenchymal stem cells and chondrocytes were collected and then co-cultured at aratio of 2 to 1.CCK-8 was used to evaluate the proliferation of the cells;Western blot was used to evaluate the expression of the related proteins.The protocol was approved by the medical ethical committee of Shanghai Jiao Tong University School of Medicin.SPSS16.0 software was used for statistical analysis.Results 1)Successful culture the condylar chondrocytes,and Alcian blue staining,Toluidine blue staining were positive.2)After Ihh signal blocking,the proliferation of condylar chondrocytes was inhibited and the apoptosis was increased.The expression of collagen type II was decreased,while the expression of collagen type X was significantly increased.3)The co-culture group had great proliferation ability.The expression of COL2A1 and COL10A1 was increased.Conclusion 1)The condylar chondrocytes easily dedifferentiate in vitro,while P2 cell is suitable for study.2)After Ihh signal blocking,the characteristic of condylar chondrocytes was lost gradually,which indicates that Ihh has an important role in the maintenance of the characteristics and function of condylar chondrocytes.3)BMSCs can maintain the phenotype of condylar chondrocytes and promote the proliferation of chondrocytes.At the same time,the condylar cartilage can induce BMSCs to chondrogenic differentiate.