【作者】 王志伟；

【导师】 周长忍；

【作者基本信息】 暨南大学 ， 生物医学工程， 2008， 博士

【摘要】 依据蛋白和有机基质调控生物矿化的机理,通过在牙釉质表面吸附牙釉基质蛋白和接枝不同活性基团等方法,系统研究了牙釉基质蛋白和不同活性基团对牙釉质表面生物矿化的影响,为深入探讨牙釉质的再矿化以及临床应用于治疗龋病提供理论基础和实验依据。本研究主要分为两部分:一、牙釉基质蛋白对牙釉质再矿化的影响采用乙酸法提取SD大鼠的牙釉基质蛋白并通过SDS-PAGE电泳对其分离纯化。利用石英晶体微天平技术研究牙釉基质蛋白在牙釉质表面的吸附行为以及在37℃模拟人工体液中诱导牙釉质表面羟基磷灰石晶体的形成和生长的作用效果。研究结果显示,大鼠牙釉基质蛋白主要为釉原蛋白和鞘蛋白。石英晶体微天平图像数据显示,共振频率随浓度的提高而降低;吸附量随浓度的提高稳定的增长;从D-F曲线中,可以得出在20μg/mL的蛋白溶液中,吸附蛋白成膜性表现出很好的附着力和膜刚性。定性分析和形貌表征显示,吸附牙釉基质蛋白的牙釉质表面矿化层含有羟基、磷酸根和碳酸根基团。牙釉质表面微球随着矿化时间的增长而增大,晶体表现出了良好的结晶度,组成上为含有少量Na⁺和CO₃^2-取代的羟基磷灰石晶体。二、牙釉质表面功能化对牙釉质再矿化的影响通过巯基与羟基的键合作用,用水解法将HS（CH₂）_nX（-SO₃H,-PO₄H₂,-COOH,-OH,-CH₃）带有的不同活性基团接枝到牙釉质表面上形成自组装单层膜,然后浸入到37℃模拟人工体液中仿生矿化。研究结果证实了五种不同活性基团（-PO₄H₂,-SO₃H,-COOH,-OH,-CH₃）成功的接枝到牙釉质表面。除接枝甲基后的牙釉质表面表现了疏水性外,均表现了良好的亲水性。形貌表征显示,受自组装膜末端活性基团的静电控制作用,不同末端基团对羟基磷灰石晶体生长速率的影响依次为:-PO₄H₂＞-SO₃H＞-COOH＞-OH＞-CH₃;受自组装膜活性基团酸碱性的影响,带有-PO₄H₂、-SO₃H、-COOH活性基团的牙釉质表面矿化生成的晶体出现了多孔状形貌;受模拟体液中的离子类别和矿化外因的影响,晶体表面出现了若干碳酸钙方块形晶体。综上所述,通过石英晶体微天平和自组装技术,成功的将牙釉基质蛋白和不同活性基团接枝吸附到牙釉质表面。研究结果表明无论是表面吸附蛋白还是接枝活性基团都能够很好的诱导羟基磷灰石晶体成核、生长和聚集,从而在国际上首次证实了蛋白和有机基质在牙釉质矿化过程中都起到了一定的调控作用,为今后利用生物矿化法治疗龋病提供试验基础。更多还原

【Abstract】 Enamel matrix proteins（EMPs） and different active groups were deposited on the enamel surface to study enamel matrix proteins and different active groups on the impact of the enamel surface’s biomineralization by means of the mechanism of Control Biomineralization with protein and organic matrix,and it provided a theoretical foundation and experimental evidence for an in-depth study of the enamel remineralization in the treatment of dental caries.This research work is divided into two parts:Part 1.the impact of EMPs on enamel remineralizationThe EMPs of SD rats were extracted by Acetic Acid Process,separated and purified by SDS-PAGE electrophoresis.The technology of quartz crystal microbalance（QCM） was applied to research the adsorption behavior of EMPs,the formation and growth induction hydroxyapatite crystals on enamel surface in simulated body fluid（SBF） solutions at 37℃.The results showed that EMPs of rats were mainly amelogienin and aneloblastin. QCM image data showed that the frequency decreased with the increased concentration, but the adsorbed amount increased steeply with the increased concentration.It was also reflected in the D-f plot that adsorption at 20μg/mL exhibits a much more viscose outer-layer structure.Qualitative analysis and morphology characterization showed that the adsorbed EMPs biomimetic mineral layers on enamel surface contained hydroxyl, phosphate and carbonate groups.The nano-spheres on enamel surface grew with the mineralization time,and crystal showed a good degree of crystallinity and contained a small amount of Na⁺ and CO₃^2-substituted hydroxyapatite crystals.Part 2.the impact of enamel surface functionalization on enamel remineralization Various active groups HS（CH₂）_nX（-SO₃H,-PO₄H₂,-COOH,-OH,-CH₃） were deposited on enamel surface by hydrolysis method,which is based on the bonding effects of sulfhydryl and hydroxyl groups,to form self-assembled monolayers（SAMs）.Then they were soaked in SBF at 37℃for biomimetic mineralization.The results indicated that thealkanethiols were firmly deposited on the enamel surface.The treated enamel surface showed a good hydrophilic except the one treated by methyl group.Morphology characterization showed that the surface functional groups dependence on apatite formation determined were as follows:The growth rates decrease in the order -PO₄H₂＞-SO₃H＞-COOH＞-OH＞-CH₃;based on the acidic and alkaline property of active groups on the SAMs,the porous crystals were emerged on enamel surface with -PO₄H₂、-SO₃H、-COOH active groups;due to the impact of ion species in SBF and external cause of mineralization,crystal surface appeared a number of box-shaped crystals of calcium carbonate.To sum up,the EMPs and various active groups were deposited on the enamel surface by the technology of QCM and SAMs.The results showed that either the EMPs or active groups can induce the hydroxyapatite crystal,nucleation growth and aggregation.Thus it firstly confirmed that the protein and organic matrix played a key role in the regulative process of enamel mineralization,and provided an experimental basis for the treatment of caries with the method of biological mineralization in the futher.更多还原