卵黄高磷蛋白调控骨矿化机制研究

Phosvitin is a highest phosphorylation protein from egg yolk, it plays a significant role in the developing bone for chick egg embryo. However, the specific mechanisms of phosvitin in the regulation of bone mineralization have not been fully recognized. This project choose high-purity phosvitin extracted from hen egg as object of research, identify phosvitin phosphorylation sites by high-precision biological mass spectrometry, and predict the compositions of structure domain and likely active sites through bioinformatics methods. Based on above information, conduct a systematic study of the phosvitin regulating biomineralization mechanism. Establish the regulation mineralization model in vitro with collagen fibrils as main matrix, then apply the characterization methods of Raman, SEM, TEM, XRD and combine the biological enzyme technology, to investigate the effect of the phosphorylation level and structure conformation of phosvitin on biomineralization regulation, resolve protein active sites, obtain the active peptide fragments with the regulation of mineralization, and clarify its regulatory mechanism. Meanwhile, highly active osteoblasts are separated and purified from chick embryo skull, study the effect of protein on cell morphology, cell proliferation and its mineralization properties, and analyze the mechanism of synergy between protein and cell regulating mineralization. The implementation of this project, will be expected to provide innovative ideas for the development of new material by biological mineralizations and deep processing of eggs.The implementation of this project, will be expected to provide innovative ideas for the development of new material by biological mineralizations and deep processing of eggs.

卵黄高磷蛋白（phosvitin）是鸡蛋蛋黄中的一种高度磷酸化蛋白，在鸡胚骨骼发育过程中扮演重要角色，然而其发挥调控骨骼矿化的作用机制并没有得到充分认识。本项目以禽蛋中提取的高纯度phosvitin 为研究对象，利用高精度生物质谱技术鉴定其磷酸化位点，运用生物信息学方法对phosvitin结构域组成和活性位点进行预测，在此基础上对phosvitin调控生物矿化作用机制展开系统研究。以胶原纤维为基质，建立体外调控矿化模型，利用Raman、SEM、TEM和XRD等手段，联合生物酶解技术，探究phosvitin磷酸化程度和蛋白构象对调控生物矿化的影响，解析蛋白活性位点，获得具有调控矿化功能的活性多肽片段，阐明其调控机制。同时，研究蛋白对成骨细胞形态及其矿化功能的影响，分析矿化过程中蛋白调控与细胞调控的协同作用机制，项目的实施，为生物矿化新材料的开发以及禽蛋的精深加工提供创新思路。

课题以蛋黄中卵黄高磷蛋白（PV）为对象，系统分析了PV的二级结构和磷酸化位点。生物信息学分析结果显示，PV与牙本质磷蛋白的结构及特性相似，进化关系相近，表明PV可能具有与牙本质磷蛋白相似的调控生物矿化的生物功能。.研究了在仿生矿化体系中PV对磷酸钙矿物晶相转化的影响。该体系中生成的磷酸钙矿物前体相为磷酸氢钙（DCPD），通过“溶解再结晶”途径，逐渐转变为更稳定的羟基磷灰石（HAP）。PV可以极大地促进DCPD向HAP的转化，使转化过程由6 h缩短至0.5 h以内。PV参与晶相转化起始阶段。.以体外胶原蛋白膜为基质，研究了PV和PPP4在钙磷仿生矿化体系中对胶原蛋白矿化的影响。通过SEM/EDS、TEM/SAED和XRD的观测发现PV和PPP4能够调控胶原纤维内部的矿化，促进ACP向HAP的转变，且形成的磷灰石晶体在胶原纤维表面也有分布。PV调控胶原纤维的矿化的发生是一个缓慢有序的过程。而在胶原电纺仿生矿化体系中，1 mg/mL的PV也可以显著促进电纺胶原纤维中DCPD向HA转化。. 进一步研究了PV对成骨细胞的影响。Pv+β-GP诱导MC3T3-E1成骨细胞矿化效果最好，phosvitin通过上调Col-I mRNA和OCN mRNA的表达，促使其下游产物ALP表达水平增加，进而诱导矿化活动增强。构建破骨细胞模型，探究了PV对RAW264.7破骨细胞生长及功能的影响机制。发现低浓度PV对RAW264.7细胞有促进增殖抑制凋亡的作用。发现PV并非通过抑制RAW264.7细胞生长而是显著地降低TRAP的活性，进而抑制RAW264.7细胞分化成为破骨细胞。. 制备了不同磷酸化程度的PV，探索不同磷酸化程度的PV对MC3T3-E1细胞增殖、分化、矿化的影响以及可能的作用机制。结果表明PV的磷酸化是促进MC3T3-E1细胞矿化的关键因素。即高磷酸化程度的PV组的细胞增殖活力，ALP 活性、矿化节点数、BMP-2mRNA、OPG / RANKL mRNA的比值较对照组均有显著性增加。. 通过碱液处理，胰蛋白酶酶解PV，经离子交换色谱和凝胶过滤色谱分离纯化蛋白磷酸肽各组分。研究卵磷酸肽各组分对矿化效果的影响，发现其中三个片段促进效果并没有PV高，而PPP3暴露了活性区域，因此促进效果最显著。促进矿化活性区域为PV核心区域中的D1165-R1258。.