Study On The Biochemical Properties Of Type Ⅱ Collagen Isolated From Blue Shark (Prionace Glauca) Cartilage And Its Effect On Human CD4~+T Cells

Collagen is the most abundant fibrillar structural protein in animals and is widely found in the extracellular matrix（ECM）of biological tissues as its key structural fibrillar protein.Currently,28 types of collagens have been identified and described.Of these,typeⅡcollagen（CⅡ）is the main component of the ECM of articular cartilage and constitutes 90–95%of the total protein content in cartilage.Numerous studies have confirmed that CII has great potential in the treatment of rheumatoid arthritis（RA）.At present,research on the biochemical properties,molecular structure,conformational relationships and biological efficacy of CII is gaining attention,while relatively few studies have been conducted both domestically and internationally on CII isolated from large marine fish.Blue sharks（Prionace glauca）are the main bycatch target in longline fisheries and gillnet fisheries and its fins and flesh are traditional seafood.Byproducts such as skin and cartilage are produced during shark processing.Therefore,in this study,CII was extracted from blue shark cartilage,its biochemical properties were characterized and the effect of blue shark typeⅡcollagen（BSCII）on human CD4⁺T cells was investigated.Chapter 1 introduced the paradoxical dual role of CII in RA and the interaction of collagen with T cells.At the same time,the rationale for this study was described.Chapter 2 described the preparation of BSCII.Collagen was isolated from blue shark cartilage using pepsin-restricted acid hydrolysis.The yield of BSCII was approximately70.80–83.00 g per kg of cartilage（7.69%±0.61%）.The protein pattern was identified using sodium dodecyl sulfate-polyacrylamide gel electrophoresis（SDS-PAGE）.The collagen isolated from the blue shark cartilage was determined to be CII with the conformation[α₁（II）]₃ and the molecular weight of theαchain was about 140 k Da.The absence of other bands on the electrophoretic profile also proved that the extracted protein reached electrophoretic purity.The biochemical properties of BSCII were investigated in Chapter 3.The total sugar content of BSCⅡwas determined by phenol-sulphuric acid method,and the results showed that its total sugar content was（4.20±0.03）%.The microstructure of BSCⅡwas observed by scanning electron microscopy（SEM）and atomic force microscope（AFM）,which showed that the solid and solution structures of BSCⅡhad the typical fibrillar structure of collagen.The amino acid composition of BSCⅡwas analysed by amino acid analyzer and ultra performance liquid chromatography-mass spectrometry（UPLC-MS）,which showed that it contained the highest amount of glycine,accounting for approximately 35.38%of the total amino acid content.Glutamic acid,alanine,proline and hydroxyproline contents were relatively high,whereas the methionine,isoleucine,tyrosine,phenylalanine and histidine contents were relatively low,and tryptophan and cysteine were not detected.The UV scanning of BSCII showed a maximum absorption peak at 223 nm.The Fourier transform infrared（FTIR）scanning of BSCII showed that the predominant absorption peaks were in the amide band,including amide A(3304 cm^-1),amide B(2938 cm^-1),amide I(1633 cm^-1)and amide II(1633 cm^-1).The absorption peaks of amide I(1633 cm^-1),amide II(1547 cm^-1)and amide III(1238 cm^-1)were further investigated.Further analysis of the absorption peaks of the amide I band showed that BSCII contained 26.98%ofβ-sheet,35.60%ofβ-turn,37.41%of random coil and noα-helix.Circular dichroism（CD）and X-ray diffraction（XRD）results of BSCII showed that it had a complete triple helix structure.The thermal stability of BSCII was analyzed by rheometer and differential scanning calorimetry（DSC）and showed a denaturation temperature of 42°C and a melting temperature of 49°C.Chapter 4 explored the effect of BSCII on human CD4⁺T cells.BSCII and human CD4⁺T cells were labelled with iFluor^TM350 succinimidyl ester dye and Cell Mask^TMGreen plasma membrane stain,respectively,and then observed by fluorescence inverted microscopy for their binding.The results showed that BSCII could bind to human CD4⁺T cells.Next,the effect of BSCII on the growth activity of human CD4⁺T cells was examined by CCK-8 assay and the results indicated that BSCII did not promote the proliferation of human CD4⁺T cells and was not cytotoxic.Finally,the expression of apoptosis-related proteins was examined by enzyme linked immunosorbent assay（ELISA）and Western Blotting（WB）.The results showed that BSCII significantly up-regulated the expression of the apoptosis-related factor Fas（/CD95）,with the most pronounced effect at a concentration of 10μg/m L,which in turn activated the Fas-mediated apoptotic pathway and caused rigidity of human CD4⁺T cells,leading to the development of immune tolerance.Chapter 5 summarized the results obtained in this study and provided an outlook on directions worthy of further research.