具有骨密度增加作用的鱼卵唾液酸糖肽化学结构解析和作用机制研究

The water soluble sialoglycoprotein from delipidated egg proteins of fresh-water fish and sea fish were prepared, then the model of controlled enzymatic hydrolysis to obtain high bioactive fish egg sialylglycopeptide was established by combined the method of pre-osteoblastic MC3T3-E1 cells proliferation activity tracing. The high bioactive target fish egg sialylglycopeptide was purified by a combination use of ultrafiltration, gel filtration chromatography, affinity chromatography and reversed phase high-performance liquid chromatography. The structures of target fish egg sialylglycopeptide were determined by tandom mass spectrometry using electrospray ionization (ESI-MS/MS).The bone metabolism regulation and bone mineral density increasing activities of fish egg sialylglycopeptide were investigated by osteoblasts and osteoclasts in vitro and the model of osteoporosis animal in vivo. The receptor activator of nuclear factor-кB ligand (RANKL) / osteoprotegerin (OPG) expression ratio of osteoblast lineage cells, which plays an important role in regulating osteoclasts differentiation and bone resorption，is a key factor linking bone formation and resorption during bone remodelng. So the molecular mechanism of bone mineral density increasing and anti-osteoporosis effects of fish egg sialylglycopeptide were studied by evaluating the expression ratio of RANKL to OPG of osteoblasts and its involvement signaling pathway. The researches will establish a MS method for analyzing the structure of fish egg sialylglycopeptide, clarity the chemical structure of fish egg sialylglycopeptide, molecular mechanism and the relationship between its structure and anti-osteoporosis effects , and provide a solid theoretical basis for high-value application of fish egg and exploitation of new functional food in anti-osteoporosis.

以淡水鱼和海水鱼的鱼卵，制备脱脂鱼卵水溶性唾液酸糖蛋白；利用成骨前体细胞MC3T3-E1的增殖活性追踪析因，建立高活性鱼卵唾液酸糖肽可控酶解模型；采用超滤、凝胶过滤、亲和层析和反相高效液相色谱等现代分离技术对酶解产物进行分离纯化，获得高活性目标鱼卵唾液酸糖肽，利用电喷雾-质谱/质谱技术解析其化学结构；通过体外培养成骨前体细胞、破骨细胞及体内建立骨质疏松症动物模型，研究鱼卵唾液酸糖肽调节骨代谢、增加骨密度的生物活性。在此基础上，以成骨细胞RANKL/OPG的表达比率为切入点，研究鱼卵唾液酸糖肽增加骨密度、抗骨质疏松的分子作用机制，进而确定其构效关系。通过本项目的研究，将建立质谱技术解析鱼卵唾液酸糖肽化学结构的方法，阐明其化学结构、作用机制和构效关系，为鱼卵的高值化加工利用和新型抗骨质疏松症功能食品的开发提供了理论依据。

鱼类加工过程中约产生30-50%包括鱼卵在内的副产物, 鱼卵中富含糖蛋白，其对骨质疏松症是否具有调节作用及其作用机制？尚未见报道。项目的主要内容是采用活性追踪技术筛选出高活性鱼卵唾液酸糖蛋白，利用质谱技术解析其化学结构，在In vitro 和In vivo 体系中研究其对骨代谢的调控作用。取得的重要成果有：利用前成骨细胞MC3T3-E1的增殖活性追踪析因，明确了鱼卵糖蛋白中唾液酸含量与其活性呈正相关性，分别从优选的淡水鱼（鲫鱼）和海水鱼（鳕鱼）的鱼卵中获得了高活性唾液酸糖蛋白；利用质谱技术和核磁共振技术，解析了其糖链、肽链结构、确定了糖基化位点，进而解明了鲫鱼卵和鳕鱼卵唾液酸糖蛋白的结构轮廓。该两种糖蛋白的化学结构均为首次报道。利用体外培养的小鼠破骨细胞、前成骨细胞、间充质干细胞模型，首次证明了鱼卵唾液酸糖蛋白具有显著抑制破骨细胞活性，促进成骨细胞活性，促进间充质干细胞向成骨细胞分化等作用，并明晰了其构效关；利用建立的多种骨质疏松症动物模型，进一步证明了鱼卵唾液酸糖蛋白具有显著改善骨质疏松症的作用。以OPG/RANKL、β-catenin为切入点，阐明了鱼卵唾液酸糖蛋白抑制骨流失与其下调OPG/RANKL/NF-κB、OPG/RANKL/MAPK信号转导通路有关，调节骨生成与调控Wnt/β-catenin和BMP2/Smads信号转导通路有关。上述生物活性的研究结果均为首次报道。.本研究共发表核心论文42篇，其中SCI收录24篇,EI收录2篇。完成相关博士和硕士学位论文10篇，获得授权专利1项，参加学术会议35/人次，其中国际会议11/人次。.本研究填补了鱼类加工副产物综合利用、糖蛋白等研究领域的多项空白，为鱼卵的高值化开发利用提供了理论依据，对于促进海洋渔业的转型升级，推动健康中国的建设进程具有重大意义。