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

我们以前的研究发现，钙依赖性中性半胱氨酸蛋白酶（calpain）在骨关节炎（OA）和类风湿性关节炎（RA）中从滑膜和软骨组织释放到滑液中，并且在体外具有很强的蛋白聚糖酶活性。这些结果表明，钙蛋白酶在OA和RA患者软骨细胞外基质（ECM）的破坏中起重要作用。对蛋白多糖上的calpain酶切位点进行分析，发现calpin在软骨细胞外基质中的蛋白多糖核心蛋白上有多个特异性的酶切位点，且酶切位点的顺序与其他类型的蛋白多糖酶切位点不同，目前已成功制备了仅识别calpain酶切位点的单克隆抗体，并直接证明了calpain在软骨细胞外基质（ECM）的破坏中起作用。此外，我还利用特异性识别calpain降解产物的单克隆抗体来检测炎性细胞因子在牛软骨组织培养体系中的影响。作为初步研究的结果，我们研制了一株特异性识别calpain降解产物的单克隆抗体，证明calpain参与了关节炎中关节软骨的破坏。

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.