Isolation And Purification Of Sea Cucumber Peptide And Its Effect On Collagen Secretion In Nih/3T3 Cells
Keywords
Peptide Proliferation Collagen Nih/3T3 Cells Stichopus Japonicas Peptide Proliferation Collagen Nih/3T3 Cells Stichopus Japonicas
Abstract
This paper isolated and purified a sea cucumber peptide from sea cucumber, and studied its effect on NIH/3T3 cell proliferation and collagen secretion. The activity tracking method was used to separate and purify sea cucumber peptide SP12 from fresh sea cucumber through ion exchange chromatography and gel chromatography. MTT method was used to detect the proliferation effect of SP12 on NIH/3T3 cells; flow cytometry was used to detect the effect of SP12 on NIH. /3T3 cell cell cycle; use the method of hydroxyproline assay kit to detect the effect of SP12 on collagen secretion of NIH/3T3 cells; use Western Blot and RT-PCR methods to detect SP12 at the protein level and gene level Effect on the expression of type I collagen, MMP-1 and TIMP-1 in NIH/3T3 cells. The results show that SP12 can significantly increase the proliferation rate of NIH/3T3 cells, promote the transition of NIH/3T3 cells from G1 phase to S phase, and increase their collagen secretion in a dose-dependent manner in the range of 0~20 ¦Ìg/mL. sex. At both the protein and gene levels, SP12 can significantly promote the expression of type I collagen and TIMP-1, and inhibit the expression of MMP-1. This may be the mechanism by which SP12 promotes collagen secretion in NIH/3T3 cells. 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 ¦Ìg/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.
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Original research was done by Song Shuliang
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