Development of a novel therapeutic agent of bone using synthetic peptide of ameloblastin

为确定成釉蛋白N端合成肽能否作为一种新型的骨缺损再生剂，我们进行了体内外分析，并获得了以下结果.为了确定成釉蛋白片段对成骨细胞具有最活跃的生物学效应，我们检测了成骨细胞系MC 3 T3-E1在处理不同长度的成釉蛋白N-末端区域后碱性磷酸酶和钙化的活性。结果表明，在成釉蛋白N端添加16个氨基酸（VPFFPQQSGTPGMASL）的诱导效果最好.为探讨16 N在成骨细胞中的信号转导途径，观察16 N处理后MC 3 T3-E1细胞中MAPK的表达。p38蛋白磷酸化.为了识别16 N受体，我们将编码16 N的cDNA转染到MC 3 T3-E1细胞中。具有分泌序列的载体能诱导MC 3 T3-E1细胞产生较高的碱性磷酸酶活性，而不具有分泌序列的载体不能诱导碱性磷酸酶活性。因此推测16 N受体可能存在于MC 3 T3-E1细胞表面.虽然膜定位受体的鉴定尚未完成，但在MC 3 T3-E1细胞的细胞膜上检测到CD 63，并怀疑其为16N.5的潜在受体。不同骨缺损模型的体内研究表明，16 N具有促进再生的作用。这些结果表明，16 N可以作为新的骨愈合促进剂

To determine whether synthetic peptide of the N-terminal of ameloblastin as a novel regenerative agent for bore defect, we achieved in vitro and in vivo analyses and obtained the following results.1. To determine the ameloblastin fragment with the most active biological effects on osteoblastic cells, we examined activities of alkaline phosphate and calcification of osteoblastic cell line, MC3T3-E1, after treatment of various lengths of N-terminal area of ameloblastin. It was show that the highest inductive effects were seem when the cells treated with 16 amino acids (VPFFPQQSGTPGMASL) at N-terminal of ameloblastin (16N).2. To examine the signaling pathway of 16N in osreoblasts, expression of MAPK in MC3T3-E1 cells after the treatment of 16N. Phosphotylation of p38 was seen in the cells.3. To recognize the receptor of 16N, we transfected cDNA encoding 16N into MC3T3-E1cells. A vector with the sequence of secretion could induce high alkaline phosphatase activity in MC3T3-E1 cells, while a vector without the sequence of secretion didn't induced. It was, therefore speculated that the receptor of 16N may present on the surface of MC3T3-E1 cells.4. Although identification of the membrane located receptor was not accomplished, CD63was detected on the cell membrane of MC3T3-E1 cells and suspected as a potential receptor of 16N.5. In vivo studies with different bone defect models showed regeneration promoting effects on 16N. These findings indicate that 16N can be used for novel agent promoter of bone healing