猪小肠黏膜下层脱细胞基质中Ⅰ、Ⅲ型胶原检测方法研究  

Study on the detection method of type Ⅰ/Ⅲ collagen in porcine small intestinal submucosa extracellular matrix

作　　者：张扬 陈毅 高建萍[2] 夏磊磊 李勇超[3] 王清石 张贵锋[2] 赵博 ZHANG Yang;CHEN Yi;GAO Jian-ping;XIA Lei-lei;LI Yong-chao;WANG Qing-shi;ZHANG Gui-feng;ZHAO Bo(Beijing Biosis Healing Biological Technology Co.,Ltd.,Beijing 100026;State Key Laboratory of Biochemical Engineering,Institute of Process Engineering,CAS,Beijing 100190;School of Life Science and Technology,Henan Institute of Science and Technology,Xinxiang 453003,China)

机构地区：[1]北京博辉瑞进生物科技有限公司,北京100026 [2]中国科学院过程工程研究所生化工程国家重点实验室,北京100190 [3]河南科技学院生命科技学院,新乡453003

出　　处：《生物学杂志》2020年第5期108-111,共4页Journal of Biology

基　　金：中关村科技园区管理委员会“重大前沿原创技术成果转化和产业化”;北京市科技新星计划(XX2018008);国家重点研发计划(2018YFA0108200);国家重点研发计划(2018YFC1106400);广东省自然科学基金项目(2014A030312013);海口市海洋经济创新发展示范城市产业链协调创新类项目(HHCL201804)。

摘　　要：猪小肠黏膜下层脱细胞基质(SⅠS,Small intestinal submucosa)的主要成分为Ⅰ、Ⅲ型胶原,拟建立一种基于液相质谱联用(HPLC-MS)的胶原类型识别和定量检测方法。首先利用胰蛋白酶将胶原标准品和样品酶解,利用离子阱质谱(Ⅰon trap mass spectrometry, LCQ)识别出Ⅰ、Ⅲ型胶原的特征多肽分别为GETGPAGPAGPVGPVGAR、GPPGAVGPSGPR,再利用三重四极杆质谱(Triple quadrupole mass spectrometry, TSQ)对其进行定量;然后通过胶原标准品浓度和峰面积的线性关系计算Ⅰ、Ⅲ型胶原的含量。结果表明VⅠDASⅠSTM和Biodesign■中Ⅰ、Ⅲ型胶原的含量总和与高效液相色谱法检测结果一致,通过HPLC-MS检测猪跟腱和真皮中Ⅰ、Ⅲ型胶原含量的结果符合其在组织中的分布规律;方法学实验表明HPLC-MS的精密度(RSD=2.38%)较高、稳定性良好。Type Ⅰ and Ⅲ collagen are the major components of porcine small intestine submucosal extracellular matrix(SIS).This study intends to establish a collagen type identification and quantitative detection method based on liquid chromatography-mass spectrometry.Firstly,the ion trap mass spectrometry(LCQ)was used to detect the digestion of the collagen standard and SIS products that had been marketing to identify the type of collagen and its marker peptides.The marker peptide of type Ⅰ collagen was GETGPAGPAGPVGPVGAR,and type Ⅲ was GPPGAVGPSGPR.The triple quadrupole mass spectrometry(TSQ)was used to detect the marker peptides,and then the contents of type Ⅰ and Ⅲ collagen in SIS were calculated by the linear relationship between the concentration and the peak area.The result showed that the contents of Ⅰ and Ⅲ in VIDASIS TM and Biodesign were close to that of the total collagen content.the contents of Ⅰ and Ⅲ collagen in porcine Achilles tendon and dermis were detected by HPLC-MS,and the results accorded with the distribution of type Ⅰ and Ⅲ collagen in tissues.

关 键 词：猪小肠黏膜下层 脱细胞基质 Ⅰ型胶原 Ⅲ型胶原 液质联用 

分 类 号：Q51[生物学—生物化学] R318.08[医药卫生—生物医学工程]