PRODUCTION OF SPECIFIC MONOCLONAL ANTIBODY FOR CALPAIN CLEAVAGE SITES AND CLINICAL APPLICATION

我们以前的报道表明，在骨性关节炎(OA)和类风湿关节炎(RA)中，钙依赖的中性半胱氨酸蛋白酶(Calain)从滑膜和软骨组织释放到滑液中，并在体外具有很强的蛋白聚糖酶活性。这些发现表明，在骨性关节炎和类风湿性关节炎患者中，钙蛋白酶在软骨细胞外基质(ECM)的破坏中起着重要作用。结果通过对蛋白多糖的分析，我们发现Calpin在关节软骨细胞外基质中的蛋白多糖核心蛋白上有许多特异的切割位点，并且在切割位置的顺序上与其他类型的蛋白多糖不同。现在我们成功地制备了一种单抗，它只识别蛋白多糖核心蛋白上的特定切割位点，并直接证明了Calain在破坏软骨的细胞外基质(ECM)中起到了作用。此外，我还使用了特异性识别Calain降解产物的单抗来检测炎症细胞因子在牛软骨组织培养体系中的影响随着炎症细胞因子的增加，细胞内释放的钙蛋白酶及其裂解的碎片量增加。我们研制了一种能特异性识别钙蛋白酶降解产物的单抗，证明了钙蛋白酶参与了关节炎关节软骨的破坏。

Our previous reports revealed that calcium-dependent neutral cystein proteinase (calpain) is released from synovial and cartilage tissue to synovial fluid in osteoarthritis (OA) and rheumatoid arthritis (RA), and has strong proteoglycanase activity in vitro. These findings suggest that calpain plays an important role in destruction of the extra-cellular matrix (ECM) of cartilage in OA and RA suffers. Result of analyzing calpain cleavage sites on proteoglycan, we detected that calpin has many specific cleavage sites on proteoglycan core protein within articular cartilage ECM and in order of the cleavage sites sequences are different from another kinds of proteoglycanases sites.Now we succeeded in production of monoclonal antibody which recognized only specific cleavage site on proteoglycan core protein by calpain, and demonstrated directly that the calain plays a role in destruction of the extra-cellular matrix (ECM) of cartilage.Furthermore, I used the monoclonal antibody which specifically recognized degradation product by calpain to examine influence of inflammatory cytokine in bovine cartilaginous tissue culture system.As a result of the pilot study, the quantity of calpain and the quantity of fragment by calpain cleavage were released from intracellular to the extracellular by adding inflammatory cytokine increased.We developed one of the monoclonal antibody which specifically recognized degradation product of calpain and proved that calpain participated in articular cartilage destruction in arthritis.