牙本质基质蛋白-1仿生多肽的设计与评价

Design and evaluation of a kind of biomimetic peptides of dentin matrix protein-1

目的设计一种牙本质基质蛋白-1(DMP-1)的仿生多肽,仿生DMP-1与胶原纤维结合,并启动矿化功能。方法依据DMP-1与胶原纤维结合的功能序列以及釉原蛋白介导羟磷灰石成核的功能序列,采用固相合成法合成DMP-1仿生多肽,其氨基酸序列为DSESSEEDRTKREEVD。利用免疫荧光法评价该多肽与胶原蛋白的结合能力;将其依次浸入氯化钙溶液和磷酸氢二钠溶液中,采用扫描电子显微镜(SEM)和透射电子显微镜(TEM)以及选区电子衍射环(SAD)分析其诱导羟磷灰石晶体成核生长的矿化能力。结果免疫荧光结果表明:本研究设计的DMP-1仿生多肽可以与牙本质胶原纤维结合;SEM、TEM观察结果显示:该多肽可以从溶液中摄取钙磷离子,诱导羟磷灰石晶体成核矿化。结论多肽DSESSEEDRTKREEVD可以模拟DMP-1与胶原蛋白结合及启动矿化的能力,可以作为研究牙本质仿生矿化的一种分子工具。

Objective To design a kind of biomimetic polypeptide of dentin matrix protein-1 （DMP-1）, which can bind to dentine collagen fibers and initiate mineralization. Methods A novel polypeptide was developed by connecting the collagen binding domain of DMP-1 ＂DSESSEEDR＂ to the hydrophilic C-terminal of amelogenin ＂TKREEVD＂. The polypeptide was synthetically prepared by standard solid-phase peptide synthesis. Human dentine slices were completely demineralized by hydrochloric acid to expose the dentine collagen. Fluorescein isothiocyanate coupled polypeptide was applied to the exposed dentine collagen. Fluorescent microscopy was used to examine the polypeptide specially bond to the dentine collagen. Nucleation and growth of hydroxyapatite was initiated by immersing the polypeptide into cal- cium chloride and sodium hypophosphate solutions respectively. Scanning electron microscopy （SEM）, transmission elec- tron microscopy （TEM） and selected area diffraction （SAD） were used to examine the hydroxyapatites formed. Results Fluorescent dentine collagen was identified in the demineralized dentine specimens. Nucleation and growth of crystals were detected after immersing the polypeptide into calcium chloride and sodium hypophosphate solutions by SEM and TEM. SAD confirmed the crystals were hydroxyapatites. Conclusion The polypeptide of ＂DSESSEEDRTKREEVD＂ can simulate DMP-1 binding collagen and initiate hydroxyapatite nucleation and growth. It may be a potential molecular tool for dentine remineralization.