胶原自组装行为的研究

发布时间：2018-04-02 22:39

本文选题：胶原　切入点：自组装　出处：《郑州大学》2017年硕士论文

【摘要】：胶原是人体内最丰富、最复杂的蛋白质,在体内通过自组装形成具有D带特征的纤维结构,是人体各种组织的重要成分。胶原蛋白性能优异,具有良好的细胞粘附性、生物相容性和生物可降解性等,特别是其自组装行为,使其成为材料科学、生物医学和生物传感器等领域独具优势和潜能的材料。然而,人们对胶原的自组装及其机理却知之甚少。本文分别采用酸法和酶法从猪皮中提取酸法胶原(ASC)和酶法胶原(PSC)。分别采用聚丙烯酰胺凝胶电泳(SDS-PAGE)、红外光谱(FTIR)、紫外光谱(UV)、差示扫描量热法(DSC)、Zeta电位和扫描电子显微镜(SEM)对所提取胶原的结构与形貌进行研究。研究发现,ASC和PSC的紫外图谱和红外图谱相似,均出现了胶原的特征峰,表明两种方法所提取的胶原均为I型胶原蛋白,且都具有三股螺旋结构;电泳图谱中ASC的β链强度高于PSC,α链强度低于PSC,且DSC曲线图中,ASC和PSC的吸热峰所对应的温度分别为56.7°C、49.2°C,说明ASC的热变性温度高于PSC,可能是ASC分子内部交联程度高于PSC。ASC和PSC的等电点分别为7.7和7.6,比较接近。SEM结果显示,冷冻干燥后的ASC、PSC均呈现膜状结构。本文系统研究了胶原浓度、外界条件(时间、温度、pH和离子强度)对ASC和PSC自组装行为的影响,分析了ASC和PSC在相同条件下的自组装行为,初步探索了胶原自组装的机理。首先,将胶原溶解于醋酸溶液中,得到胶原溶液,然后使其在磷酸缓冲液(PBS)中自组装。采用UV、流变仪和SEM等手段表征分析了胶原的自组装行为。研究发现,胶原自组装过程可分为成核和纤维生长两个阶段。随着时间的延长,胶原纤维逐渐形成,D带也越来越明显。当胶原浓度高于100μg/mL时,胶原能发生自组装;调节胶原浓度,可以调节胶原自组装的速率和纤维的密集度。当温度低于4°C时,观察不到明显的自组装行为;升高温度可以加快胶原自组装的速率。但是,当温度高于50°C时,胶原发生变性,失去自组装能力。流变数据显示:胶原自组装过程是可逆的,但在解组装过程中,胶原并未完全解自组装成胶原分子,可能是形成了细小的纤维。pH、离子强度会对胶原自组装动力学和形态结构产生明显的影响。在等电点附近和一定离子强度范围内(200 mM-300 mM),有利于胶原自组装行为的发生。ASC和PSC自组装后的形貌结构没有明显区别,但在动力学方面,ASC的自组装速率快于PSC,可能是由于端肽引起的,说明端肽可以加快自组装速率,而对形貌结构几乎没有影响。在探索胶原自组装机理的基础上,进一步研究了大分子对胶原自组装行为的影响,为通过自组装制备胶原基复合材料的研究提供理论支持。
[Abstract]:Collagen is the richest and most complex protein in human body.Collagen has excellent cell adhesion, biocompatibility and biodegradability, especially its self-assembly behavior, which makes it a promising material in the fields of material science, biomedicine and biosensor.However, little is known about the self-assembly of collagen and its mechanism.In this paper, acid method and enzymatic method were used to extract acid collagen and enzymatic collagen from pig skin respectively.The structure and morphology of the extracted collagen were studied by polyacrylamide gel electrophoresis (page) SDS-PAGEN, FTIR, UV, DSCC Zeta potential and scanning electron microscope (SEM), respectively. The structure and morphology of the extracted collagen were studied by scanning electron microscopy (SEM) and differential scanning calorimetry (DSC). The structure and morphology of the extracted collagen were studied by scanning electron microscopy (SEM) and differential scanning calorimetry (DSC).It was found that the ultraviolet and infrared spectra of ASC and PSC were similar and had the characteristic peak of collagen, which indicated that the collagen extracted by the two methods was type I collagen and had three helical structure.The 尾 chain strength and 伪 chain strength of ASC were higher than those of PSC, and the endothermic peaks of DSC and PSC were 56.7 掳C and 49.2 掳C, respectively, indicating that the thermal denaturation temperature of ASC was higher than that of ASC, and the degree of internal crosslinking of ASC might be higher than that of PSC.ASC.The isoelectric points of PSC and PSC were 7.7 and 7.6, respectively.After freeze drying, ASCPSC all showed membrane structure.In this paper, the effects of collagen concentration, external conditions (time, temperature, pH and ionic strength) on the self-assembly behavior of ASC and PSC were systematically studied. The self-assembly behavior of ASC and PSC under the same conditions was analyzed, and the mechanism of collagen self-assembly was preliminarily explored.Firstly, collagen was dissolved in acetic acid solution to obtain collagen solution and then self-assembled in phosphoric acid buffer solution (PBS).The self-assembly behavior of collagen was characterized by UV, rheometer and SEM.It was found that the process of collagen self-assembly can be divided into two stages: nucleation and fiber growth.With the extension of time, the formation of D-band of collagen fibers became more and more obvious.When collagen concentration is higher than 100 渭 g/mL, collagen self-assembly can occur, and the rate of collagen self-assembly and fiber density can be adjusted by adjusting collagen concentration.When the temperature is below 4 掳C, no obvious self-assembly behavior is observed, and the rate of collagen self-assembly can be accelerated by increasing the temperature.However, when the temperature is higher than 50 掳C, collagen denaturation and loss of self-assembly ability.The rheological data showed that the process of collagen self-assembly was reversible, but the collagen was not completely self-assembled into collagen molecules during the unassembly process.It is possible that the formation of fine fibers, pH, ionic strength will have a significant effect on the self-assembly kinetics and morphology of collagen.In the vicinity of isoelectric point and in a certain range of ionic strength, the morphology and structure of collagen self-assembly. ASC and PSC after self-assembly have no obvious difference.However, the self-assembly rate of ASC is faster than that of PSC in kinetic aspect, which may be due to the terminal peptide, which indicates that the terminal peptide can accelerate the self-assembly rate, but has little effect on the morphology and structure.On the basis of exploring the mechanism of collagen self-assembly, the effects of macromolecules on collagen self-assembly behavior were further studied, which provided theoretical support for the preparation of collagen-matrix composites by self-assembly.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O629.73

【相似文献】