海参肽的分离纯化及其对NIH/3T3细胞胶原蛋白分泌的影响  

Isolation and Purification of Sea Cucumber Peptide and Its Impact on the Secretion of Collagen by NIH/3T3 Cells

作　　者：宋淑亮[1] 王欢[1] 梁浩[1] 吉爱国[1,2] SONG Shu-liang WANG Huan LIANG Haol JI Ai-guo(Marine College, Shandong University Weihai, Weihai 264209, China School of Pharmaceutical Sciences, Shandong University, Jinan 250012, China)

机构地区：[1]山东大学(威海)海洋学院,山东威海264209 [2]山东大学药学院,山东济南250012

出　　处：《现代食品科技》2017年第3期22-28,共7页Modern Food Science and Technology

基　　金：国家自然科学基金资助项目(81371455)

摘　　要：本论文从刺参中分离纯化得到一种海参肽,并研究其对NIH/3T3细胞增殖和胶原蛋白分泌的影响。采用活性追踪的方法从新鲜刺参中通过离子交换层析、凝胶层析分离纯化得到海参肽SP12,采用MTT法检测SP12对NIH/3T3细胞的增殖作用;采用流式细胞术检测SP12对NIH/3T3细胞细胞周期的影响;采用羟脯氨酸测定试剂盒的方法检测SP12对NIH/3T3细胞胶原蛋白分泌的影响;采用Western Blot及RT-PCR的方法,在蛋白水平及基因水平上检测SP12对NIH/3T3细胞I型胶原蛋白、MMP-1及TIMP-1表达的影响。结果表明SP12能显著提高NIH/3T3细胞的增殖率,能促进NIH/3T3细胞由G1期向S期转变,增加其胶原蛋白的分泌量,且在0~20μg/mL范围内均呈剂量依赖性。在蛋白水平和基因水平,SP12均能显著促进I型胶原蛋白及TIMP-1的表达,抑制MMP-1的表达,这可能是SP12促进NIH/3T3细胞胶原蛋白分泌的作用机制。A sea cucumber peptide, SP12, was isolated and purified from Stichopus japonicus, and its impact on NIH/3T3 cell proliferation and secretion of type I collagen was studied. SP12 was purified by ion exchange chromatography and gel filtration chromatography from fresh Stichopus japonicus using a cell activity tracking method. The effect of SP12 on NIH/3T3 cell proliferation was determined by 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide（MTT） assay, and its impact on cell cycle progression was determined by flow cytometry. The effect of SP12 on collagen secretion by NIH/3T3 cells was measured by hydroxyproline kit, and its impact on the expression of type I collagen, matrix metalloproteinase-1（MMP-1）, and tissue inhibitor of metalloproteinases-1（TIMP-1） in NIH/3T3 cells was measured at the protein and gene levels by western blot and reverse-transcription polymerase chain reaction（RT-PCR）. The results clearly demonstrate that SP12 can considerably enhance the proliferation rate of NIH/3T3 cells and promote progression from G1 phase to S phase. In a range of 0~20 μg/mL, SP12 enhanced collagen secretion by NIH/3T3 cells in a dose-dependent manner. At the protein and genetic levels, SP12 considerably promoted the expression of type I collagen and TIMP-1, and inhibited the expression of MMP-1; this might be the mechanism underlying SP12 promotion of collagen secretion by NIH/3T3 cells.

关 键 词：多肽 增殖 胶原蛋白 NIH/3T3细胞 刺参 

分 类 号：TS201.2[轻工技术与工程—食品科学]