一种釉质特异性生物矿化模板的构建

Construction of a specific biomineralization template of enamel

目的构建一种自组装类釉原蛋白两亲性寡肽,作为釉质仿生矿化的有机模板。方法依据釉原蛋白分子在釉质形成中自组装成"纳米球"超分子结构的机制,参照目前"自组装多肽类生物材料"的设计特点,通过对釉原蛋白分子功能性残基进行改性,构建"类釉原蛋白两亲性寡肽"生物矿化模板,采用固相合成法合成并纯化,经质谱仪、高效液相色谱仪等鉴定。在寡肽溶液中加入1 mol/L的CaCl2溶液观察其能否自组装;将组装后的寡肽涂于透射电镜的铜网上,用2.5%的戊二醛固定1 h后先置于常温或40℃下10 mmol/L的CaCl2溶液中1 h,去离子水漂洗后再置于5 mmol/L的Na2HPO4溶液中1 h,5个循环后取出自然干燥,通过扫描电镜(SEM),透射电镜(TEM)及选区电子衍射(SAEDS)观察表征。结果类釉原蛋白两亲性寡肽序列为C18H35O-Thr-Lys-Arg-Glu-Glu-Val-Asp,高效液相色谱仪(HPLC)示多肽纯度为98.29%,质谱仪(MS)示肽的分子质量为1 142.41 u。通过Ca2+可使其自组装为纳米纤维超微结构的凝胶态。通过交替矿化发现组装后的类釉原蛋白两亲性寡肽能摄取溶液中的钙磷离子,在寡肽纳米纤维表面成核矿化,形成磷灰石晶体。结论成功构建了一种类釉原蛋白两亲性寡肽,可以作为釉质仿生的特异性生物矿化模板,用于诱导釉质再矿化研究。

Objective Construct a self-assembly amelogenin-inspired oligopeptide amphiphile as organic matrix template for simula- ting mineralization of enamel. Methods Inspired by amelogenin that self-assembles into nanospherical supermolecular structures in enamels and according to the design features of ＂self-assembly polypeptide biological materials＂ ,a novel biomineralization template of ＂amelogenin-like oligopeptide amphiphile＂ was designed be modify the funtions of amelogenin. The oligopeptide amphiphile was synthe- sized by standard solid-phase peptide synthesis, purified and characterized by high performance liquid chromatography （HPLC） and mass spectrometry （MS）. Peptide self-assembly was induced with 1 mol/L CaC12 solution to see whether it can self assemble. Ca^2＋ -in- duced self-assembled peptides were smeared onto a holey TEM copper grid and fixed with 2.5% glutaraldehyde at room temperature for 1 h. Grids loaded with self-assembled peptide were immersed in 10 mmol/L CaC12 solution for one hour at room temperature or 40 22 in oven and then rinsed in deionized water. The samples were then exposed to 5 mmol/L Na2 HPO4 solution as did in CaC12 solution fol- lowed by rinsing. This cycle was repeated five times, after which the specimens were removed, dried in atmospheric conditions, and ex- amined with SEM,TEM and selected area electron diffraction （SAED）. Results The sequence of the amelogenin-inspired oligopep- tide amphiphile was ClsH350-Thr-Lys-Arg-Glu-Glu-Val-Asp. Peptide molecule weight （MV） and purity were 1 142.41 and 98.29% accordingly,identified with MS and HPLC respectively. The oligopeptide amphiphile could self-assemble into a gel in nanofiber ultra mi- crostructure by Ca^2＋ induction. It was observed after alternative mineralization that peptide fiber could adsorb calcium and phosphate i- ons from the solution. Then, minerals crystals nucieated, grew on the surfaces of the peptide fibers, and orientated along the fibers. Con- clusions A novel amelogenin-inspired oligopeptide amphiphile was designed and synthesized, which could be a specific template for biomimetic mineralization of enamel, and could be useful in the study of inducing enamels＇ remineralization.