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

下一步，用免疫组织化学方法检测GADD45β在人和动物关节软骨中的表达，其中GADD45β在人早期网状软骨中高表达。软骨细胞肥大在骨性关节炎动物模型中呈阳性反应，而在正常动物中不表达。这些结果提示GADD45β可能参与了软骨细胞去分化的机制。为了从软骨细胞表型的角度检测GADD45β的机械功能，采用软骨细胞系进行了荧光素酶活性测定和实时荧光定量聚合酶链式反应。有趣的是，当GADD45β过表达时，X型胶原和基质金属蛋白酶-13基因的表达增加，而通过siRNA抑制GADD45β后，X型胶原和基质金属蛋白酶-13基因的表达降低。此外，GADD45β过表达使II型胶原基因表达降低，GADD45β抑制使II型胶原基因表达增加。我们还利用ATDC5细胞进行了细胞凋亡分析。GADD45beta具有明显的抗细胞凋亡作用，可促进X型胶原和基质金属蛋白酶-13基因的表达，抑制II型胶原基因的表达。这些结果提示GADD45β在骨性关节炎中的重要作用，在骨性关节炎过程中支持软骨细胞的存活，导致软骨细胞与正常软骨细胞去分化。

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.