| At present,most collagen comes from land mammals such as cattle,sheep,and pigs,but the use of mammalian collagen is limited due to mad cow disease and religious factors.Therefore,it is of far-reaching significance to find new biologically-derived collagen resources.There is a large amount of aquatic product waste on fishery products,such as fish skin and fish bone.Type ? collagen is related to autoimmune diseases and has immune activity.So we studied terrestrial type ? collagen(Chicken)and fish type ? collagen(Andrias davidianus and Tilapia).Analyzed the differences in the immune effects of collagens from different sources on the immune effects of T lymphocytes.The molecular composition of collagen is linked to its biological activity.Analyze the differences in molecular structure and biological activity of collagen from different biological sources.Type ? collagen from Chicken?Andrias davidianus and Tilapia were isolated and purified.Analysis and comparison of amino acid composition and structure of collagen.Compare the biochemical structures of three collagens.To study the structural differences of three biologically derived collagens and their role in in vitro immune activity.The first chapter describes the structure of type ? collagen and its biochemical characteristics due to its special structure,the mechanism of oral immune tolerance,and the relationship between type ? collagen and autoimmune diseases,and the effect of oral type ? collagen in animal experiments and clinical research.The second chapter introduces the extraction and purification of Chicken type ? collagen,Andrias davidianus type ? collagen and Tilapia type ? collagen.Isolation of type ? collagen by acid-limiting pepsin method.Dodecyl sulfate polyacrylamide electrophoresis gel(SDS-PAGE)was used to analyze the electrophoresis patterns of three type ? collagens and determine the molecular weights of the subunits.The SDS-PAGE spectra showed that the electrophoretic bands of the three type II collagens were similar,and all of them consisted of three identical ?1 chains,and the molecular weight of the ?1 chain was about 130 k Da.The third chapter introduces the amino acid composition of Chicken type ? collagen,Andrias davidianus type ? collagen and Tilapia type ? collagen,analyzes the amino acid composition characteristics of the three type ? collagens,and compares the differences.The percentage of total amino acid residues in the type ? collagen sub-amino acids(proline and hydroxyproline)is 14.6%.Compared with the other two collagens,its content is the highest and its degree of hydroxylation is higher.The content of methionine and leucine in the hydrophobic amino acids of Tilapia type ? collagen was significantly higher than that of the other two types of collagen ? Glycine,glutamic acid,and arginine are high in the pharmacodynamic amino acids of collagen,and the type ? collagen pharmacodynamic amino acids(glutamic acid,glycine,and arginine)of Andrias davidianus,Tilapia,and Chicken account for 47.8%,46.9%,and 46.4% respectively,of which the highest effective amino acid residue content of Andrias davidianus type ? collagen.Terrestrial-derived collagen is less active than fish collage.The fourth chapter analyzes the biochemical characteristics of Chicken type ? collagen,Andrias davidianus type ? collagen and Tilapia type ? collagen.The thermal degradation characteristics of type ? collagen were analyzed by scanning calorimetry and thermogravimetric analysis.Fourier transform infrared spectroscopy was used to analyze the infrared spectral structural characteristics of collagen.Scanning electron microscope and atomic force microscope were used to observe the microstructure of collagen.The results of differential scanning calorimetry showed that the thermal denaturation temperatures of big cockles and chickens were 30.43 and 30.46 ?,which were lower than the tilapia fish collagen 31.04 ?.The weight loss of the three type II collagens is divided into two stages.The first stage is the loss of free water and partially bound water.The second stage is the destruction of amino acid residues in the collagen peptide chain,which results in deamination and dehydration.Fourier infrared spectra showed that all three type ? collagens had preserved the complete triple helix Structure.Scanning electron microscopy showed that the Tilapia type II collagen fibers were tightly and orderly arranged.The fifth chapter to study the Chicken ? type collagen,Andrias davidianus type ? collagen and Tilapia ? collagen type immune active in vitro.Taking human leukemia lymphoblast MOLT-4 as the research object,cck-8 method was used to analyze the toxic effects of three collagens on MOLT-4 cells.The effects of three collagen-treated cells on TNF-?,IL-6,and IL-1? secretion were measured by ELISA.The results showed that the three collagens had different secretion of cytokines.Tilapia type ? collagen down-regulated TNF-? secretion.Chicken type ? collagen up-regulated IL-6 secretion.All type II collagens down-regulated IL-1? secretion.Both Andrias davidianus and Chicken type ? collagen promoted the secretion of apoptosis-related factor Fas/Apo-1.Compared with the control group,when the collagen concentration was 100 ?g/m L,Fas/Apo-1 secretion level was highest in the Andrias davidianus and Chicken type ? collagen treatment group.When the Tilapia type ? collagen concentration was 25 ?g / m L,compared with the control group,the content of Fas/Apo-1 secreted by MOLT-4 cells was significantly different(P <0.01).The three collagens can promote the apoptosis level of MOLT-4 cells,but they vary with the concentration of collagen.The main reason may be related to the degree of hydroxylation of collagen from different biological sources.In chapter 6,the effect of three collagens on the expression of apoptotic cytokines in MOLT-4 cells were studied.RT-PCR was used to determine the expression of cytokines by three type ? collagens.All three type ? collagens increased gene expression of the apoptotic factors Fas/Apo-1,Caspase-3 and Caspase-8 and the expression of inflammatory factors interleukin-6(IL-6)and interleukin-2(IL-2),Compared with the control group,the gene expression level of interleukin-10(IL-10)was the highest when tilapia type ? collagen concentration was 25?g/m L.The three type ? collagens can promote the apoptosis of MOLT-4 cells by activating the Fas signal channel and the Caspase cascade reaction pathway,and through gene regulation,promote the expression of intracellular cytokines,resulting in down-regulation of the immune response.The amino acid composition of collagen from three different biological sources is different.The degree of hydroxylation of fish collagen is higher than that of chicken collagen.The content of hydrophobic amino acids in tilapia collagen is higher than the other two collagens.This is the main molecular structure difference of the three collagens.The degree of hydroxylation of type ? collagen affects the immune tolerance of collagen as an antigen.Hydrophobic amino acids are also associated with the interaction between antigens and antibodies.In general,collagen type ? induces MOLT-4 cell apoptosis,promotes the release of cytokines,and can cause immune tolerance in vitro. |
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