Pathological mechanism of osteoarthritis -analysis of molecules responsing to mechanical stress-

下一步，使用免疫组织化学方法检测人源和动物关节软骨中gadd45 β的表达。GADD45beta在网状软骨早期OA软骨簇中表达特别高。骨性关节炎动物模型软骨细胞增生性阳性，正常动物无阳性。这些发现提示gadd45 β在骨性关节炎过程中支持软骨细胞去分化的机制。为了检测gadd45 β在软骨细胞表型方面的机械功能，我们利用软骨细胞系进行荧光素酶测定和实时荧光定量PCR。有趣的是，过表达GADD45beta会增加X型胶原蛋白和MMP-13基因的表达，但使用siRNA抑制GADD45beta会降低表达。II型胶原基因表达通过GADD45beta过表达而降低，通过GADD45beta抑制而升高。我们还研究了ATDC5细胞的凋亡分析。GADD45beta具有明显的抗凋亡作用。该蛋白加速X型胶原蛋白和MMP-13基因表达，抑制II型胶原蛋白基因表达。gadd45 β在体外抗软骨细胞凋亡方面也具有良好的功能。这些结果提示gadd45 β在OA过程中支持软骨细胞存活的重要性，从而导致正常软骨细胞的去分化。

In the next step, the expression of GADD45beta was examined using immunohistochemistry of articular cartilage from human and animal samples.GADD45beta was especially highly expressed in cluster of early OA cartilage in human reticular cartilage. The hypertrophic chondrocyte were positive in OA animal model but not in normal animal. These findings suggested GADD45beta functions to support the mechanism of dedifferentitation of chondrocyte during the process of OA.To examine the mechanical function of GADD45beta in terms of phenotype of chondrocyte, luciferase assay and real time PCR were performed using chondrocyte cell line. Interestingly, type X collagen and MMP-13 gene expressions were increased by GADD45beta over-expression, but decreased by GADD45beta suppressing using siRNA. Additionally, type II collagen gene expression was decreased by by GADD45beta over-expression and increased by by GADD45beta suppressing. We also investigated apoptosis analysis using ATDC5 cells. GADD45beta clearly functioned anti-apoptotic way.This protein accelerates Type X collagen and MMP-13 gene expression and suppresses Type II collagen gene expression. GAdd45beta also noble function in terms of anti-apoptosis in chondrocyte in vitro.These results suggest the importance of GADD45beta function in OA to support the survival of chondrocytes during the process of OA, resulting in dedifferentiation from normal chondrocyte.