基于唾液蛋白质组学的仿生防龋功能多肽构建及其应用基础研究

Human saliva is one important body fluid containing proteins and peptides which have the potential of anticaries.The acquired enamel pellicle (AEP) is a thin acellular film that forms on tooth surfaces upon exposure to the oral environment which consists predominantly of salivary proteins. The saliva and AEP plays a key role in the maintenance of oral health by regulating processes including demineralization, remineralization and shaping the composition of early microbial flora adhering to tooth surfaces.Many proteins in saliva and AEP function through maintaining the dynamic equilibrium between demineralization and remineralization,some other proteins may contribute to the defense against microorganisms.This highlights that salivary proteins can be used for anticaries applications. However, the translation of potential salivary anticaries functional peptides into clinical settings is hampered by the dynamics and complexity of the salivary proteome. Recently, technological developments and achievements relating to salivary proteomics make it possible to characterize salivary protein composition and investigate its impact on development of dental caries.By means of salivary proteomics, this project aims to create biomimetic anticaries functional peptides out of proteins which are sifted from saliva and AEP.This can be assumed as the most advanced idea to create novel biomimetic anticaries peptides. In a first step,high-throughput analysis will be used to generate accurate protein/peptide profile.  The  differential  proteins between the salivary proteome of caries-active patients and caries-free controls will be analyzed for the purpose of selecting proteins and peptides with the ability to prevent and treat dental caries. Molecular biotechnology will then be used to determined the structure of the potent anticaries proteins and peptides, a series of novel biomimetic anticaries peptides will be designed and synthesized by mimicing the functional domain of these proteins and peptides.Use of stability assay and cytotoxicity assay will allow the selection of several promising biomimetic anti-caries peptides with stable structure in oral conditions and minimal hemolytic activity and cytotoxicity.The anticaries activity of these biomimetic anticaries will be evaluated with the intent of selecting biomimetic functional peptides with remarkable control of cariogenic bacteria and efficient ability to promote remineralization and inhibit demineralization of initial caries lesions.Finally,in order to promote the clinical application of the biomimetic anticaries functional peptides,the parameters of peptides delivery will be systematically optimized. This project is possessing a high potential to revolutionize the application of salivary proteomics in the field of prevention and treatment of dental caries,exploitsing a new land of creating biomimetic anticaries peptides, and establishing a complete set of evaluation system of anticaries activity in vitro and in vivo. Meanwhile, this is the first time to take in vivo environment into consideration at all stages of design, sifting and activity evaluation of anticaries peptides. Bionic anticaries peptides from saliva and enamel acquired pellicle possess preferable adaptability in oral conditions, relative to our previous odontogenic biomimetic anticaries peptides, and therefore implementation of this project will significantly stimulate the improvement of clinical anticaries efficacy of biomimetic anticaries peptides, and lay a solid groundwork for the biomimetic anticaries approach, which is new, safe and in possession of the independent intellectual property rights.

唾液及获得性膜中存在天然防龋蛋白。借助唾液蛋白质组学技术，筛选并仿生唾液天然防龋蛋白可能成为构建仿生防龋功能多肽的理想新模式，迄今国内外尚无此方面研究。本项目创新性筛选唾液及牙面获得性膜中天然防龋蛋白成分，分析并借鉴其结构，合成唾液仿生防龋功能多肽。通过多肽结构稳定性及安全性评估、抗菌防龋活性筛选实验、矿化防龋活性筛选实验，构建结构稳定、安全性高且效果显著的唾液仿生防龋功能多肽，并对其应用模型进行研究。本项目建立了唾液蛋白组学在龋病防治领域的新应用，将其从龋病诊断、监测领域拓展到防龋因子研发领域。本项目开拓了防龋功能多肽的仿生新领域，来源于唾液蛋白的仿生防龋多肽比前期牙源性仿生防龋多肽具有更好的口腔环境适应性。因此，本项目将仿生防龋功能多肽的研究推向更接近临床的高度，项目的实施将显著提高仿生防龋功能多肽临床应用效果及可能性，为仿生防龋这一安全有效的具有自主知识产权的防龋途径研发奠定基础。

本项目针对龋病发病率高、防治不佳的现状，创新性提出基于唾液和获得性膜蛋白质组分析设计获得结构稳定、安全性高且效果显著的唾液仿生防龋功能多肽用于龋病防治的研究理念。本项目采用iTRAQ联合MRM技术验证得到高龋组与无龋组人群唾液差异蛋白136种，其中具有潜在防龋功能的蛋白包括histatin1，statherin, mucin-7, mucin-5B，basic salivary proline rich protein-2（PRB-2）等。采用Label-free鉴定得到龋易感和无龋人群获得性膜的差异蛋白100种，其中IgG、IgA、lysozyme C、mucin-7等差异蛋白被PRM进一步验证。生物信息学分析发现低龋组唾液上调蛋白多属于细胞外空间和免疫系统过程蛋白，而获得性膜差异蛋白主要与宿主免疫反应和抗菌活性有关。根据差异蛋白间的统计学差异和差异倍数确定目标唾液蛋白为statherin, histatin1, PRB-2，设计合成了3条唾液蛋白促矿化防龋功能多肽，分别是statherin衍生的DK6和DE11，histatin1衍生的DK5，PRB-2蛋白衍生的PG12。基于乳铁蛋白N端抗菌序列，设计合成乳铁蛋白衍生防龋抗菌肽LF-1和LF-2。以上多肽经HPLC-MS鉴定合成成功，且没有明显的细胞毒性和溶血性。多肽DE11、DK5、PG12对牙釉质表面具有良好的吸附能力，能够促进钙磷离子成核，经多肽处理早期釉质龋再矿化后表面显微硬度恢复率高于阴性对照组，龋损深度和矿物丢失量显著低于阴性对照组。此外，多肽处理组的大鼠磨牙残存釉质体积和密度均高于阴性对照组，平滑面龋和窝沟龋数明显低于阴性对照组。另一方面，LF-1对变异链球菌和放线菌具有较强的抗菌活性，而LF-2对大多数实验菌株的抗菌活性均高于LF-1，两者局部使用均可明显降低大鼠Keyes指数。最后，以脂质体作为唾液蛋白促矿化防龋功能多肽的应用模型制备DK-5脂质体，并完成DK5脂质体的表征、体外释放、稳定性和安全性评估。体外及体内实验分析表明DK-5脂质体具有良好的早期釉质龋促矿化效应和生物安全性。本课题是基于唾液及获得性膜天然防龋蛋白构建口腔环境内防龋作用稳定的仿生功能多肽新思想的首次提出，建立了一套包括蛋白筛选、多肽稳定性、安全性、防龋有效性的防龋活性研究体系，切实提高仿生防龋功能多肽临床应用的可能性。