Gene Expression Analyses of Subchondral Bone in Early Experimental Osteoarthritis by Microarray

Osteoarthritis (OA) is a complex degenerative joint disease, which is not only a cartilage but also a bone disease. A better understanding of the early molecular mechanism changes of subchondral bone in vivo may contribute to elucidating the pathogenesis of OA. We used microarray technology to investigate the time-course molecular changes of subchondral bone just beneath damaged cartilage in early stage of experimental osteoarthritis, and found 2,234 differentially expressed (DE) genes at 1 week, 1,944 at 2 weeks and 1,517 at 4 weeks postsurgery.Further analysis of dysregulated genes indicated that subchondral bone remodeling occurred sequentially and in a time-dependent manner at the gene expression level. Some known dysregulated genes suspected roles in influencing bone development or bone remodeling, such as Alp, Igf1, Tgf ß1, Postn, Mmp3, Tnfsf11, Acp5, Bmp5, Aspn and Ihh, were confirmed by real-time PCR, and results indicated that our microarray data could accurately reflect gene expression patterns of early OA. Subsequently, to validate the results of our microarray analysis at protein level, immunohistochemistry staining was introduced to investigate the translational level of genes Mmp3 and Aspn in tissue sections, and results showed that the level of Mmp3 protein expression was totally matched the results of microarray and real-time PCR analysis. Nevertheless, the expression of Aspn protein was not observed differentially expressed at any time point.骨关节炎（Osteoarthritis, OA）是一种复杂的退行性关节疾病，其病变不仅累及软骨，同时也属于骨骼疾病范畴。深入阐明体内软骨下骨的早期分子机制变化，或可为揭示骨关节炎的发病机制提供重要依据。本研究采用微阵列（microarray）技术，探究实验性骨关节炎早期阶段受损软骨下方的软骨下骨的时序性分子改变，发现术后1周、2周及4周分别存在2234个、1944个和1517个差异表达（differentially expressed, DE）基因。对失调基因的进一步分析显示，在基因表达层面，软骨下骨重塑呈现时序性且时间依赖性的变化模式。部分已被推测参与骨发育或骨重塑的已知失调基因，如Alp、Igf1、Tgf ß1、Postn、Mmp3、Tnfsf11、Acp5、Bmp5、Aspn及Ihh，经实时荧光定量PCR（real-time PCR）验证，结果证实本研究的微阵列数据可准确反映早期骨关节炎的基因表达特征。随后，为在蛋白水平验证微阵列分析结果，本研究采用免疫组织化学染色（immunohistochemistry staining）技术，检测组织切片中Mmp3与Aspn基因的翻译水平，结果显示Mmp3蛋白的表达水平与微阵列及实时荧光定量PCR的分析结果完全吻合。然而，在所有检测时间点均未观察到Aspn蛋白存在差异表达。