环氧化酶-2控制骨骼发育与疾病中软骨细胞成熟与肥大新机制的研究

The overall objective of this project is to define a novel Cox-2-related regulatory mechanism during chondrocyte hypertrophy. Chondrocyte hypertrophy and its specific marker, the type X collagen gene (Col10a1), are known to play essential roles in endochondral ossification during skeletal development. Mutations and abnormal expression of human COL10A1 are often accompanied by abnormal chondrocyte hypertrophy as found in many skeletal disorders. Therefore, molecular regulators that govern cell-specific Col10a1 expression are very likely the candidate therapeutic targets for many skeletal diseases. We have previously demonstrated that Runx2 interacts with Col10a1 cis-enhancer and is an indispensible transcription factor for Col10a1 expression. More recently, we identified Cox-2 as a novel candidate Col10a1 regulator. This is intriguing, as Cox-2 (cyclooxygenase) is an enzyme that is involved in formation of the important prostanoids. Functionally, pharmacological inhibition of COX-2 can provide relief from the symptoms of inflammation and pain. Cox-2 is upregulated in various carcinomas suggesting a role in tumorigenesis. Interestingly, Cox-2 has also been shown to play essential roles during skeletal repair and positively regulates chondrocyte hypertrophic differentiation. However, the genetic evidence and molecular mechanism of Cox-2 regulation of chondrocyte hypertrophy are still lacking. Here, we surmise that "there is a novel Cox-2/Runx2-centered Col10a1 regulatory mechanism that plays essential roles in chondrocyte hypertrophy during endochondral bone formation". To test this, we propose: Aim #1 to determine the molecular mechanism by which Cox-2 regulates Col10a1 expression; Aim #2 to determine how Cox-2 affects chondrocyte hypertrophy during skeletal development and disease; and Aim #3 to identify Col10a1 regulators during chondrocyte hypertrophy in vitro.

本研究旨在阐明环氧化酶-2（Cox-2）调节软骨细胞成熟的新机制。软骨细胞成熟及其标志基因，COL10A1，在软骨内成骨中起重要作用。该基因变异多伴发软骨细胞成熟受损并常见于多种骨骼疾病。因此，COL10A1基因调节因子是相关骨骼疾病的治疗靶点。先前的研究表明Runx2与Col10a1基因增强子结合，是其表达必需的转录因子。我们最近的研究发现Cox-2是Col10a1基因新的调节因子。已知Cox-2参与前列腺素合成，与炎症、疼痛及肿瘤发生相关。有意义的是， Cox-2还与骨修复和软骨细胞成熟有关，但缺乏遗传学证据。我们推测："以Cox-2/Runx2 为中心的Col10a1基因调节机制在软骨细胞成熟时起重要作用"。为此，我们的目标是：1， 研究Cox-2如何调节Col10a1；2， 确立Cox-2如何影响软骨细胞成熟；3，应用软骨细胞成熟模型分离鉴定Col10a1基因调节因子。

本项目最初的立题依据是，期望通过研究Cox-2对软骨细胞成熟的标志基因，Col10a1基因的表达调控，从而阐明Cox-2如何调节软骨细胞的成熟分化及相关机制。为此，我们的研究设定了3个目标：1， 研究Cox-2如何调节Col10a1；2， 确立Cox-2如何影响软骨细胞成熟；3，应用软骨细胞成熟模型分离体鉴定Col10a1基因调节因子。四年多来，我们很好地完成或超额完成了上述指标，相关的内容已分别发表于多篇1、2和3区专业论文、研究生论文和在此前的进展报告中也有汇报，具体分述如下：.1）：我们的研究表明Cox-2促进软骨细胞肥大与成熟分化，并且与Runx2等多种转录因子一起构成调节Col10a1基因表达的新型机制，进而影响骨骼发育与疾病中软骨细胞成熟的进程。该部分研究成果已经发表（Oncotarget. 2016）。.2）：为了寻找与Runx2共同调节软骨细胞成熟肥大的因子，我们以Runx2 为基础诱饵进行了酵母双杂交饵并筛选源自肥大性软骨细胞的cDNA文库。我们成功地筛选到30多个候选因子，包括 Lectin-1 (Lgals1), Col1a2, Edf1 and Timp-2等。生物信息学与初步的表达分析表明这些候选因子在增值与肥大性软骨细胞中呈差异表达。该部分研究内容也已发表（Am J Transl Res. 2016）。.3）：我们还探讨了miRNA-101表观遗传控制Col10a1基因表达和软骨发育成熟以及相关疾病的影响。我们的研究结果表明miR-101在肥大性MCT 细胞较增值性MCT 细胞的相对表达量减低。转染 miR-101类似物可以降低肥大性MCT细胞中Col10a1的mRNA 水平，而抑制miR-101可见Col10a1的mRNA 水平升高，说明miR-101对于Col10a1的负性调控作用。我们还应用荧光定量PCR和Western blot检测了转染miR-101类似以及抑制片段后相关标志基因的相对表达量，但结果呈多样性。相关的研究内容已被Am J Transl Res接收，待发表。四年来，本课题组在该项目或与该项目相关的领域已发表论文10余篇，待发表论文6-8篇，均为通讯或共同通讯作者.