大豆粘性功能肽的构效关系、粘性机理及对骨折愈合作用的研究

Safe, efficient, non-toxic medical bio-adhesive peptide from soy protein has important social and economic significance. However, how to prepare such peptides with high-added value from soy protein, and its structure-activity relationships (SAR) are unknown, and its adhesive mechanisms are not yet fully elucidated. In the present study, highly adhesive peptides from the subunits of soybean protein will be obtained by using a novel limited proteolysis and separation. The shear and tensile strengths of adhesive peptides will be investigated using both in vitro and in vivo evaluation methods, and their adhesive structures will be characterized using functional proteomics, circular dichroism (CD), infrared spectroscopy (2D-IR), differential scanning calorimetry (DSC) and so on. In addition, we will further confirm the SAR and characterization of adhesive peptides, identify the functional regions for affecting its adhesive strength, and clarify the adhesive molecular mechanism using replacement (mutation) of amino acid method and by determining of the correlations between adsorption, infiltration, and adhesion properties of highly adhesive peptides with the substrate (animal bones). Moreover, we will incubate the osteoblasts (MC3T3-E1) cells with adhesive peptides and determine the effect of its peptides on the proliferation, differentiation, and safety of cells. We will establish the rabbit models of mandibular fractures and glue by the adhesive peptides, and then observe ossification and perform biological evaluation during the process of fracture healing. Taken together, this study will have significant implications in understanding important SAR and molecular mechanisms of adhesive peptides in the process of fracture healing, and provide insightful theoretical basis for developing the high-value processing and utilization of soybean protein as new medical bio-adhesives.

从大豆蛋白中制备安全、高效、无毒的医用粘性肽具有重要的社会和经济意义，而阐明其构效关系和粘接作用机制是目前亟待解决的问题。本研究将采用独创的限制酶解技术，以在体和离体动物骨骼为实验对象进行粘接性能的评价，从大豆亚基蛋白中筛选出高粘接活性的功能肽；用质谱技术、圆二色光谱（CD）、红外光谱（2D-IR）、差示扫描量热法（DSC）等方法对高粘接活性肽进行结构鉴定与表征，运用氨基酸变异技术对粘性肽进行构效关系和表征的研究，确定其粘性作用的功能区域（关键位点），在分子水平阐明其粘性机理；通过粘性肽与动物骨骼的吸附、浸润和粘附性能的研究，阐明其粘接作用机理；以成骨细胞（MC3T3-E1）和动物骨折模型为研究对象，采用多种研究方法和角度从不同水平阐明粘性肽在骨折愈合中的作用。本研究将阐明大豆粘性肽的构效关系、粘接机理及其在骨折愈合中的作用，为大豆蛋白高值化加工利用和新型医用生物粘合剂的开发奠定理论基础。

以大豆蛋白为原料制备安全、高效、无毒的医用胶粘剂具有重要的社会和经济意义。主要研究内容和重要结果的包括：（1）以粘合强度为指标，研究了大豆分离蛋白（SPI）胶的pH、浓度、固化时间对猪扇骨粘合性能的影响，并通过正交实验优化了其具有最佳粘合性能的工艺参数。研究表明SPI胶对猪扇骨具有最佳粘合性能的参数为：浓度10%，pH 6.5，固化时间48h，此时的粘合强度达到380KPa；（2）以体外动物骨骼为试验对象，研究了羧甲基纤维素钠（CMC-Na）对SPI胶粘合性能的影响。研究表明CMC-Na的添加，导致SPI胶二级结构的改变。α-螺旋含量降低而β-折叠含量增加，表明蛋白质分子展开程度增加，内部疏水基团暴露，表面疏水性提高，增加了SPI胶零切黏度，从而显著提高低浓度SPI胶的骨粘合强度，而低浓度SPI胶更利于机体吸收；（3）以体外动物骨骼为试验对象，研究了瓜尔豆胶（Guar gum, GG）、黄原胶（Xanthan gum, XH）及瓜尔豆胶与黄原胶1:1（GG/XH）比例混合形成的复配胶对SPI胶粘合性能的影响。研究表明多糖的添加使得SPI胶中各组分氢键相互作用增强，α-螺旋百分含量降低，蛋白分子结构无序性增强，蛋白分子结构趋于柔软松散，从而生成无定形活性结构，零切粘度增大，很好改善了SPI胶与骨片之间粘合性能。添加GG/XH的SPI胶的粘合强度达到698KPa；（4）通过酪氨酸酶修饰大豆蛋白中的酪氨酸生成多巴胺可实现大豆蛋白的改性；（5）通过两步酶解SPI，并以超滤、葡聚糖凝胶柱分离等手段对酶解产物进行分离纯化，以成骨细胞MC3T3-E1为体外细胞模型，筛选出了对成骨细胞的增殖和分化具有促进作用的大豆粘性肽；（6）进行了可能与骨折愈合相关的成骨细胞分化（Osteoblast differentiation）信号通路的研究，阐明了IGF-I/IGFBP2刺激Vimentin/RPTP调控成骨细胞分化与生长的作用机制，探究了大豆粘性蛋白（肽）对骨骼粘接作用及其在骨折方面应用的可能。因此，本研究课题阐明了大豆蛋白胶的粘接机理及其在骨折愈合中的作用，为大豆蛋白高值化加工利用和新型医用生物粘合剂的开发奠定理论基础。