Searches for the molecular pathogenesis of skeretal and neuronal system from serum calcium-decreasing factor, caldecrin, knock out mouse

钙化蛋白是从胰腺中分离和克隆的一种血清降钙因子，可能是一种急性胰腺炎的降钙因子。它属于弹性蛋白酶家族，但其降钙活性与蛋白水解酶活性没有明显的联系。我们发现钙化蛋白的降钙活性抑制了破骨细胞性骨吸收和骨髓破骨细胞的形成。我们还发现，在RANKL诱导的破骨细胞形成的信号转导通路中，Caldecrin抑制了钙离子-钙调神经磷酸酶系统。另一方面，钙化蛋白在大脑中表达，其生理功能尚不清楚。我们研究了钙化蛋白是否抑制了阿尔茨海默病老年斑块中淀粉样蛋白的形成。Caldecrin以时间依赖的方式消化淀粉样蛋白β多肽40和42，并抑制淀粉样蛋白β多肽42的形成。此外，这些消化的多肽片段的神经毒性也消失了。这些结果表明，在大脑中表达的钙解蛋白消化淀粉样蛋白β，并通过蛋白酶活性抑制淀粉样蛋白的形成。因此，我们制作了钙化蛋白基因破坏小鼠，以探索其在骨骼和神经系统中的生理和病理功能。将钙蛋白基因的靶向载体重组到C57BL/6胚胎干细胞中，筛选出G418抗性克隆。通过主成分分析和Southern印迹杂交分析，获得了多个克隆。

Caldecrin is a serum calcium-decreasing factor which was purified and cloned from pancreas, and seems to be a hypocalcemic factor with acute pancreatitis. It belongs to the elastase family, but its calcium-decreasing activity did not clear connect with protease activity. We identified that the calcium-decreasing activity of caldecrin suppressed osteoclastic bone resorption and osteoclast formation from bone marrow. We also identified that caldecrin suppressed Ca ion-calcineurin system in the signal transduction pathway of the RANKL-induced osteoclstic formation. On the other hand, caldecrin expresses in the brain, and its physiological function is not clear. We examined whether caldecrin suppressed an amyloid formation in the senile plaque of Alzheimer's disease. Caldecrin digested amyloid β peptides 40 and 42 with time-dependent manner, and also suppressed amyloid formation of amyloid, β peptide 42. In addition, neurotoxicity of these digested peptide fragments was disappeared. These results suggest that caldecrin expressed in the brain digests amyloid β and suppresses amyloid formation by protease activity. Therefore we made caldecrin gene destruction mouse for the search of physiological and pathological functions in the bone and nervous system. A targeting vector for caldecrin gene was recombinated into an C57BL/6 embryonic stem cell and selected G418-resistant clones. We obtained several clones analyzed by PCA and southern blot hybridization.