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

我们合成了一种模拟骨细胞黏附蛋白骨涎蛋白(BSP)功能序列的多肽。BSP模型肽(E_7PRGDY)由BSP的一个羟基磷灰石结合序列(E_7)和一个细胞附着序列(RGD)组成，与人工合成的羟基磷灰石晶体具有亲和力。这些细胞附着在包有BSP或多肽的晶体上，尽管它们没有附着在包有血清白蛋白的晶体上。贴附在包被多肽的晶体上的细胞被压扁并形成伪足。过量的RGD多肽可抑制细胞的附着。该多肽可以通过聚(GIU)序列连接到晶体上，并将RGD序列暴露在介质中。细胞表面的整合素可以与RGD序列相互作用。我们还检测了破骨细胞与包裹着蛋白质的晶体的附着。通过向小鼠骨髓细胞分化制备破骨细胞。骨髓细胞在含1，25(OH)_2D_3的晶体上培养，抗酒石酸酸性磷酸酶染色检测破骨细胞。观察到破骨细胞附着在包被BSP或BSP模型肽的晶体上。过量的RGD多肽抑制了这种附着，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附着，其多聚(Glu)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.