Identification of inhibitor of osteoclastogenesis

本研究的目的是纯化和鉴定从小鼠基质细胞（前成骨细胞，TMS-12）中释放的破骨细胞形成抑制剂。为了定量测定破骨细胞形成抑制剂的活性，我们建立了破骨细胞形成的测定系统，并确定了无血清培养条件下TMS-14的培养条件，以纯化抑制剂。利用该系统，我们用离子交换、分子切割、透析等方法对120升TMS-14条件培养基中的抑制剂进行了纯化，最后用Superdex 200 HR 10/30测定了抑制剂的分子量为200 K和22 K。现在，我们正在克隆这些物质。接下来，我们对抑制剂进行了表征。结果，抑制剂的特征如下：1）通过抗体中和实验确定这些抑制剂不是已知的破骨细胞形成抑制剂IL-4、IL-10、TNFα; 2）这些物质抑制破骨细胞分化，但不抑制破骨细胞祖细胞增殖，第三章这些物质还改变了成熟破骨细胞的形态，抑制了破骨细胞形成窝的形成，这些数据表明，破骨细胞形成抑制剂的特性与其他抑制剂有很大的不同如骨保护蛋白/破骨细胞生成抑制因子。

The purpose of this research was to purify and identify inhibitors of osteoclast formation released from mouse stromal cells (pre-osteoblasts, TMS-12). To quantify the activity of inhibition of osteoclast formation, we developed the assay system for osteoclast formation, and to purify the inhibitors we determined the cultured condition of TMS-14 without serum. Using this system, we carried out the purification of inhibitors from the 120 liter of the conditioned medium of TMS-14 with ion-exchangers, molecular cutting, dialysis and so on. Finally after applying Superdex 200HR10/30 it was determined that the molecular of the inhibitors was 200K and 22K. Now, we carry out the cloning of this substances. Next, we characterized the inhibitors. As a result, the inhibitors were characterized as follows ; 1) It was determined the inhibitors was not IL-4, IL-10, TNFα, known-inhibitors of osteoclast formation by the experiments of antibody neutrization, 2) The substances inhibited osteoclast differentiation, but not proliferation of osteoclast progenitor cells, 3) The substances also changed the morphology of mature osteoclasts and inhibited the pit formation.These data indicated that the characterization of the inhibitors of osteoclast formation was quietly different from other inhibitors such as osteoprotegrin/osteoclastogenesis inhibitory factor.