软骨特异性表达rAd-col2A1-shRNA-ADAM8/EGFR治疗骨关节炎的实验研究

The key of arthrodial cartilage destruction of osteoarthritis was more degradation and less synthesis of extracellular matrix (ECM). Previous studies showed one of mechanisms by which ECM was degraded and regulated. The ligands, including HB-EGF and TGF-α, were hydrolyzedfrom cell surface by active ADAM8. Then the ligand combined with EGFR receptor on the cell surface and the ras-raf-erk/p38MAPK signaling pathway was activated while the expression of MMPs increased. In our study, we intended to build over-expression vector of ADAM8/EGFRto observe its effects on ECM degradation and adeno-associated virus-mediated chondrocyte-specific expression vector, named rAd-col2A1-mir30-shRNA-ADAM8/EGFR to interfere up-regulation of ADAM8/EGFR. Then key transcriptional factors in degradation of cartilage ECM and the expression of specific function gene were detected. We expect to explore molecular mechanisms of regulation of cartilage matrix degradation by ADAM8 and EGFR and observe its value on treating osteoarthritis to provide theoretical basis for clinical treatment of osteoarthritis and other cartilage ECM lost diseases.

骨关节炎关节软骨被破坏的核心是细胞外基质的降解超过了合成。研究发现，软骨细胞外基质的降解调节机制之一可能是通过有活性的ADAM8水解细胞表面的配体，包括HB-EGF和TGF-α，同细胞表面的受体EGFR结合激活ras-raf-erk/p38MAPK通路，进而上调MMPs的表达来实现的。本课题拟构建ADAM8/EGFR过表达载体转染软骨细胞，观察其对ECM降解的影响；构建软骨细胞特异性表达腺相关病毒介导的rAd-col2A1-mir30-shRNA- ADAM8/EGFR，分别对上调的ADAM8/EGFR予以RNA干扰，对软骨细胞外基质降解关键转录因子和特异性功能基因的表达进行检测，探索ADAM8和EGFR调控软骨细胞外基质降解转录水平的分子机制,观察治疗骨性关节炎的价值，为骨性关节炎及其他软骨细胞外基质丢失疾病的临床治疗提供理论依据。

骨关节炎（OA）是最常见的关节疾病之一，迄今为止尚无有效治疗药物或方法。软骨细胞外基质（ECM）的降解是OA的主要原因。研究发现，软骨细胞外基质的降解调节机制之一可能是通过有活性的ADAM8 水解细胞表面的配体，包括HB-EGF 和TGF-α，同细胞表面的受体EGFR 结合激活ras-raf-erk/p38MAPK 通路，进而上调MMPs 的表达来实现的。首先，建立大鼠原代软骨细胞系和IL-1β诱导的软骨细胞炎症模型。过表达或敲减ADAM8基因表达后，检测EGFR 介导的EGFR / ERK / NF-κB通路相关基因的表达。同时，探讨Notch信号通路通过Hes1与ADAM8的直接相互作用正反馈调节ECM降解的分子机制。结果显示骨关节炎软骨细胞中ADAM8的表达上调。ADAM8的敲减抑制了体外OA细胞模型中的OA表型。ADAM8通过激活EGFR/ERK/NF-κB信号通路来调节OA进展。抑制Notch信号传导会抑制体外OA细胞模型中的OA表型。Notch信号通过Hes1直接调节ADAM8的基因表达。Notch1-ADAM8正反馈环促进体内OA的进展。结论：Notch1-ADAM8反馈环调节软骨细胞外基质的降解和骨关节炎的进展。本课题探索性研究了Notch1-ADAM8反馈环调控软骨细胞外基质降解转录水平的分子机制,探讨其治疗骨性关节炎的价值，为骨性关节炎及其他软骨细胞外基质丢失疾病的临床治疗提供理论依据。