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蛋白调控骨、牙矿化的研究及模拟
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摘要
本文采用材料学分析、表征等手段,结合生物学相关技术,对两种骨、牙基因变异模型中矿物结构和性能进行了分析,探讨了基因、细胞和蛋白变化对矿化过程的影响。在此基础上设计了两种多肽片段,分别模拟非胶原蛋白和釉原蛋白的结构和功能,研究了其对I型胶原矿化和对晶体生长组装的影响,试图从分子水平上探讨生物矿化过程中的蛋白调控机制。
     col1-caPPR型鼠牙釉质中,出现了片状晶体等异常形态;其排列方式紊乱;结晶性和成熟度明显下降;釉柱组装散乱;力学性能明显下降。这些变化表明,釉原蛋白等的紊乱表达影响了晶体的形核、生长及组装,成釉细胞的分化削弱、排列紊乱破坏了釉柱、釉间质紧密结构。本文首次报道了在牙本质内异常发育的牙釉质,证实了釉牙本质界存在成釉细胞和成牙本质细胞之间的信号传导。
     Axin2 KO型鼠骨中,成骨细胞数量增加,活性提高;胶原纤维分泌旺盛,排列组装更加有序;新生骨小梁中胶原纤维更易于形成板层结构;皮质骨变厚,板层结构变薄,板层数量增加,结构更加密实;矿物结晶度和成熟度都有所提高。表明Axin2 KO鼠骨中骨更新更加有效,使皮质骨纳米力学性能明显增加。
     本文所设计的多肽片段(EEEEEEEEDSESSEEDR)在钙离子作用下呈β-折叠,促进了HA晶体的定向生长和平行排列;通过静电作用结合于胶原e1区,促进了其纤维化过程,增加了形核位点,促进了矿物沉积和晶型转变;促进了牙本质的修复再矿化。该多肽片段有望应用于骨组织工程和牙本质修复等领域。
     本文建立了牙釉质腐蚀早期脱矿模型,其腐蚀最先从有机鞘开始,釉柱区域腐蚀较快而釉间质晶体较晚被腐蚀;最终形成完全脱矿层、软化层、过渡层和正常层等多层结构。该定量观测方法可应用于饮料、牙膏和临床研究等领域。
     本文设计的高分子—多肽(DMPA-PCL-P)能够自组装形成200 nm左右的纳米球结构。该自组装结构能够影响HA晶体的平行排列组装,促进相邻晶体间融合、生长。这种结构有助于我们理解釉原蛋白的调控作用,也有助于探索新型的有机大分子模板用于调控无机晶体生长。
In the present study, the mineral structure and mechanical properties of enamel and bone from two transgenic mice models were investigated by combining the materials characterization and analysis methods with the traditional biological techniques. Based on these results, the impacts of gene, cells as well as proteins on their mineralization processes were discussed from nano-scale to macro-scale. Then two different peptide fragments (or polymer peptide complex) were designed to simulate the structures and functions of noncollagenous proteins or amelogenin. Their regulations on the mineralization of collagen as well as on the arrangement and growth of hydroxyapatite were analyzed, which provided some important information about the control mechanism of protein on biomineralization from the molecular level.
     The disturbed enamel biomineralization in col1-caPPR mice was investigated for the first time in this study. The SEM results displayed abnormal enamel distribution and altered crystal arrangement in transgenic enamel, especially the prism organization without decussation and the loosely packing crystals, which were significantly different from wild-type. The FTIR investigations revealed poorer crystallinty and maturity in transgenic enamel. The loosely packing crystals and abnormal prism organization resulted in inferior mechanical properties. All these differences indicated that the heterogeneous expressions of amelogenin and ameloblastin as well as the impaired differentiation and disorganization of ameloblasts altered the biomineralization process during enamel development in col1-caPPR mice.
     Femurs from the 6- and 12-month-old female Axin2 KO mice were investigated compared with normal controls. Osteoblast proliferation and differentiation were significantly increased with increased expression and more ordered arrangement of collagen fibrils. The collagen fibrils in newly formed trabecular preferred to organize in lamellar pattern rather than randomly. The trabecular number and thickness increased significantly, while the trabecular resembled plate-like morphology compared to the rod-like morphology in wild-type mice. Also, the cortical bone became thicker with more thinner lamellar. In addition, their mineral became more mature and crystalline with less substituted ions, the nanohardness and elastic modulus were significantly increased.
     The peptide fragment (EEEEEEEEDSESSEEDR) showedβ-sheet structure in the presence of Ca2+. The formation ofβ-sheet could be an ideal template to control the HA crystals grow in preferred orientation and arrange parallel to each other along their c-axis. It was likely that peptide was preferentially bound to the e-band of type I collagen (the hole zones) by electrostatic interactions. The adsorption of peptide on the collagen influenced the assembly of collagen fibrils in vitro, and also increased the fibril diameter. The peptide-collagen interaction promoted the mineralization of collagen and demineralized dentin by providing more nucleation sites.
     A multilayer structure characterizing the early stage of enamel erosion was established based on AFM, SEM and nanoindentation. This model could be used to measure the amount of the enamel loss directly, and to describe the softened layer and transition layer qualitatively or quantitatively. The approach presented here may be useful in clinical surgery, also the early stages of food inducing native enamel dissolution could potentially be measured.
     The polymer peptide complex (DMPA-PCL-P) could self-asssemble to form globular aggregates of nearly 200 nm in diameter in vitro. These nanospheres facilitated preferred growth and fusion between the adjacent hydroxyapatite crystals. The designed structure would be helpful for us to understand the regulation of amelogenin on mineralization, also provide a reference for designing new organic templates to induce and control crystal growth and arrangement.
引文
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