鲫鱼卵唾液酸糖蛋白化学结构分析及对前成骨细胞MC3T3-E1增殖分化的影响

目的：成熟的鲫鱼卵经提取、分离纯化得到唾液酸糖蛋白（sialyglycoproteins of Carassius auratus eggs，CA-SGP），分析其化学结构，及对前成骨细胞MC3T3-E1增殖分化的影响。方法：采用水提法从成熟鲫鱼卵中提取水溶性蛋白，经阴离子交换色谱、凝胶过滤色谱分离纯化鲫鱼卵水溶性蛋白得到CA-SGP，测定其基本组成，采用高效凝胶液相色谱和聚丙烯酰胺凝胶电泳测定其分子质量和纯度，PMP柱前衍生高效液相色谱法测定其单糖组成；采用噻唑蓝法测定MC3T3-E1细胞的增殖率，酶联免疫法测定Ⅰ型胶原蛋白（collagen type Ⅰ，COLⅠ）和骨钙素（osteocalcin，OCN）的分泌量及碱性磷酸酶（alkaline phosphatase，ALP）的活性，茜素红染色法测定矿化结节的数量。结果：得到单一的组分CA-SGP，在高效凝胶过滤色谱色谱图上呈现单一的糖和蛋白的检测重叠峰，在凝胶电泳图上呈现单一弥散性条带，其蛋白质含量为14.33%，己糖含量为62.81%，N-乙酰神经氨酸（N-acetylneurainic acid，Neu5Ac）含量为19.72%。单糖组成测定结果显示：CA-SGP主要由甘露糖、葡萄糖胺、半乳糖胺组成，其物质的量比为7.61∶6.70∶1.00；MC3T3-E1细胞与CA-SGP共同孵育后，其增殖率显著增加，COLⅠ和OCN的分泌量分别增加了39.28%和83.06%，ALP活性提高了285.08%，矿化结节的数量增加了96.41%，说明CA-SGP显著促进MC3T3-E1细胞增殖、分化和矿化。结论：CA-SGP为富含Neu5Ac的糖蛋白，具有显著促进MC3T3-E1细胞增殖、分化和矿化的作用，对开发抗骨质疏松功能性食品具有重要意义。

Chemical Structure Analysis of Sialyglycoproteins of Crucian Carp Carassius auratus Eggs and the Effect of the Sialyglycoproteins on Proliferation and Differentiation of MC3T3-E1 Cells

This study was designed to investigate the chemical structure of sialyglycoproteins from crucian carp Carassius
auratus eggs (CA-SGP) and the effects of CA-SGP on the proliferation, differentiation and mineralization of MC3T3-E1
cells. CA-SGP was isolated from the water soluble protein of crucian carp eggs using anion exchange chromatography and
gel exclusion chromatography, and its composition was determined. The proliferation of MC3T3-E1 cells was determined
by MTT assay, the secretion of collagen type (COLⅠ), alkaline phosphatase (ALP), and osteocalcin (OCN) were detected
by enzyme-linked immunosorbent assay (ELISA) and the mineralized nodule of MC3T3-E1 cells was evaluated by staining
with Alizarin red S dye. The analysis of chemical structure showed that the CA-SGP was composed of 14.33% protein
and 62.81% carbohydrates in which the content of N-acetylneurainic acid (Neu5Ac) was 19.72%, and the monosaccharide
composition of CA-SGP was Man, GlcN, GalN, and Neu5Ac. The activity assays indicated that the CA-SGP effectively
promoted the proliferation of MC3T3-E1 cells, significantly increased the secretion of ALP, OCN and COLⅠand enhanced
the mineralization ability. In conclusion, CA-SGP significantly promotes the proliferation, differentiation and mineralization
of MC3T3-E1 cells and can be used as an active ingredient in a safe and effective anti-osteoporosis functional food.