猪脑来源糖胺聚糖的分离纯化及精细组成分析  

Isolation,purification and detailed structure analysis of glycosaminoglycans from porcine brain

作　　者：金鑫[1,2] 王晨 赵小亮[1,2] 李国云 于广利[1,2] JIN Xin;WANG Chen;ZHAO Xiao-liang;LI Guo-yun;YU Guang-li(Key Laboratory of Marine Drugs, Ocean University of China, Qingdao 266003,China;Shandong Provincial Key Laboratory of Glycoscience and Glycotechnology, Medicine and Pharmacy Institute of Ocean University, Qingdao 266003,China)

机构地区：[1]海洋药物教育部重点实验室中国海洋大学,青岛266003 [2]山东省糖科学与糖工程重点实验室中国海洋大学医药学院,青岛266003

出　　处：《中国新药杂志》2019年第6期726-731,共6页Chinese Journal of New Drugs

基　　金：国家自然科学基金资助项目(31600646);山东省自然科学基金资助项目(ZR2016HB42)

摘　　要：目的:从猪脑中提取分离糖胺聚糖(glycosaminoglycans,GAGs),并进行精细结构分析。方法:猪脑组织经脱脂、蛋白酶酶解后,经氯代十六烷基吡啶(CPC)富集,乙醇沉淀获得糖胺聚糖粗品。利用Q-Sepharose Fast Flow离子交换柱纯化,获得7个组分(P1～P7)。所得分级样品分别经肝素裂解酶与硫酸软骨素裂解酶酶解,2-氨基吖啶酮(AMAC)荧光标记后,利用液质联用方法分析其精细结构。结果:猪脑GAGs粗品中主要获得硫酸乙酰肝素(heparan sulfate,HS)、硫酸软骨素(chondroitin sulfate,CS)和少量透明质酸(hyaluronic acid,HA),含量分别为307,293和18 mg·kg^(-1)。P2～P4组分GAGs含量较少; P5～P7组分CS和HS含量相当,CS中以ΔUA-GalNAc4S为主; HS组成以ΔUA-GlcNAc为主,电荷密度略低,未获得肝素组分。结论:基于液质联用技术分析猪脑组织的精细结构,为动物来源糖胺聚糖的研究提供依据。Objective: To extract and purify glycosaminoglycans (GAGs) from porcine brain and analyze their fine structures. Methods: Homogenized porcine brain was defatted then digested with trypsin and papain. The crude GAGs were enriched by cetylpyridinium chloride (CPC) and precipitated by ethanol. Seven fractions were eluted by Q-Sepharose Fast Flow column and named as P1~P7, respectively. The crude GAGs and purified fractions were digested by heparin lyase Ⅰ/Ⅱ/Ⅲ and chondroitin lyase ABC , respectively. The generated disaccharides were labeled with 2-AMAC and then analyzed by LC-MS. Results: The GAGs isolated from porcine brain were mainly composed of heparan sulfate (HS) and chondroitin sulfate (CS) with trace amount of hyaluronic acid (HA), and their content were 307 , 293 and 18 mg·kg^-1 , respectively. GAGs were less in P2~P4 , and P5~P7 had similar contents of CS and HS. CS from P5~P7 were all primarily composed of △UA-GalNAc4S. And △UA-GlcNAc was the main disaccharide detected in HS, which had a lower charge density. No heparin was acquired from porcine brain. Conclusions: This study evaluated the contents and exact composition of GAGs in porcine brain based on LC-MS.Detailed characterization of the disaccharide structures and compositions is beneficial for study on animal-derived GAGs.

关 键 词：猪脑组织 分离纯化 糖胺聚糖 二糖组成 精细结构 

分 类 号：R931.6[医药卫生—生药学]