Identification of inhibitor of osteoclastogenesis

本研究的目的是纯化和鉴定小鼠基质细胞（前成骨细胞，TMS-12）释放的破骨细胞形成抑制剂。为了量化破骨细胞形成的抑制活性，我们建立了破骨细胞形成测定系统，并测定了无血清TMS-14的培养条件，以纯化抑制剂。利用该系统对120升TMS-14条件培养基进行了离子交换、分子切割、透析等纯化。最后应用Superdex 200HR10/30后，确定抑制剂的分子为200K和22K。现在，我们进行这种物质的克隆。接下来，我们对抑制剂进行了表征。因此，抑制剂的特征如下：1)通过抗体中和实验，确定该抑制剂不是已知的破骨细胞形成抑制剂IL-4、IL-10、TNFα； 2)该物质抑制破骨细胞分化，但不影响破骨细胞祖细胞的增殖；3)该物质还改变成熟破骨细胞的形态，抑制窝的形成。这些数据表明，破骨细胞形成抑制剂的特性与其他抑制剂（如骨蛋白素/破骨细胞生成抑制因子）截然不同。

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.