DESIGN OF A CELL-ATTACHMENT PEPTIDE WHICH CONTROLS CELLULAR ACTIVITY IN BONE

我们合成了一种模拟骨唾液酸蛋白（BSP）功能序列的肽，BSP是骨中的一种细胞附着蛋白。BSP模型肽（E_7PRGDY）是由BSP的羟基磷灰石结合序列（E_7）和细胞附着序列（RGD）组成的，它对人工合成的羟基磷灰石晶体具有亲和性。细胞附着在包被BSP或肽的晶体上，尽管它们不附着在包被血清白蛋白的晶体上。附着在肽包被晶体上的细胞变平并发育伪足。用过量的RGD肽抑制附着。肽可以通过聚（Giu）序列连接到晶体，并将RGD序列暴露于介质。细胞表面的整合素可以与RGD序列相互作用，我们还观察了骨细胞与蛋白质包被晶体的粘附。通过诱导小鼠骨髓细胞分化制备成骨细胞。骨髓细胞在含1，25（OH）_2D_3的培养基中培养。抗酒石酸酸性磷酸酶染色检测骨细胞。观察到骨形成细胞附着在用BSP或BSP模型肽包被的晶体上。过量的RGD肽抑制了附着，表明整合素参与了这种附着。结论：BSP可介导成骨细胞和破骨细胞在骨矿物质上的粘附，其多聚谷氨酸序列和RGD序列参与了这一功能。

We synthesized a peptide which mimics functional sequences of bone sialoprotein (BSP), a cell attachment protein in bone. This BSP model peptide (E_7PRGDY) is composed of a hydroxyapatite-binding sequence (E_7) and a cell-attachment sequence (RGD) of BSP.This peptide has affinity to synthetic hybroxyapatite crystals.We examined attachment osteoblastic MC3T3-E1 cells to the crystals coated with proteins. The cells were attached to the crystals coated with BSP or the peptide, although they were not attached to the crystals coated with serum albumin. The cells attached on the peptide-coated crystals were flattened and developed pseudopods. The attachment was inhibited with excessive amount of a RGD peptide. The peptide may be attached to the crystals through the poly (Giu) sequence and expose the RGD sequence to a medium. Integrins on the surface of the cells can interact with RGD sequence.We also examined attachment of osteoclastic cells to the crystals coated with proteins. The osteoclastic cells were prepared by differentiating murine bone marrow cells. The bone marrow cells were cultured on the crystals in a medium containing 1,25 (OH) _2D_3. Osteoclastic cells were detected by staining for tartrate-resistant acid phosphatase. Osteoclastic cells were observed attached on the crystals coated with BSP or the BSP model peptide. The attachment was inhibited with excessive amount of the RGD peptide, indicating involvement of integrins in this attachment. We conclude that BSP can mediates attachment of osteoblasts and osteoclasts on bone mineral and that its poly (Glu) sequence and RGD sequence are involved in this function.