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

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0~20 μg/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。

Abstract:

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.