【作者】 王碧；

【导师】 张铭让； 张廷有；

【作者基本信息】 四川大学 ， 皮革化学与工程， 2003， 博士

【摘要】 制革废弃物的大部分是皮边角余料，其胶原蛋白占80％以上，胶原蛋白不仅是丰富的蛋白质、氨基酸营养源，同时是优良的功能性生物高分子材料，因其特有的生物学特性，在医药、食品、功能性保健品、化妆品、农业等多个领域都具有广泛的应用前景，因此，回收利用皮边角余料中胶原蛋白资源具有重要意义。 本论文基于我国制革废弃物的应用研究现状及胶原蛋白的性能优势与缺点，在减轻制革污染的同时，变废为宝，以高值转化为目的，在对从制革皮边角余料中提取的胶原蛋白进行分析表征的基础上，围绕胶原蛋白的应用特性和共混生物膜材料进行了研究。 论文的研究内容和取得结果如下： 1．测定了提取的胶原蛋白和胶原肽的相对分子质量。建立了一种新的测定小分子多肽相对分子质量的方法，该方法用于相对分子质量小于20kD的小分子胶原蛋白和胶原肽相对分子质量测定效果较好。解决了小分子胶原蛋白和胶原肽相对分子质量测定的难题。 2．系统测定不同相对分子质量范围的胶原蛋白及多肽的物化性能表明：由制革皮边角余料提取的胶原蛋白及多肽均具有良好的应用特性，胶原蛋白及多肽溶液的粘度不仅受相对分子质量的影响，而且受浓度、温度、酸度、外加电解质和存放时间等多种因素的影响。 3．针对纯胶原应用于组织工程等领域有力学性能差，亲水性过强，在含水条件下难以塑形，不宜于内部器官的构建；体内及新生组织细胞产生的胶原酶极易使其降解，不能与组织细胞生长繁殖的速度相匹配等缺点。首次制备了胶原蛋白-葡甘聚糖(C-KGM)、胶原蛋白-葡甘聚糖-壳聚糖(CKCS)、胶原蛋制革废弃物提取的胶原特性及与多糖共混生物膜材料的研究白一葡甘聚糖一软骨素(CKCs)3种迄今未见报道的胶原蛋白一多糖新型共混生物膜。利用红外光谱(盯一IR)、X一射线衍射(X.RD)等手段表征了共混膜的结构与形态，研究了共混物的相互作用及相容性;并分别测定了共混膜的力学性能、吸水率和热稳定性等物理性能。并采用光电子能谱(抑s)和表面能测试研究了三元共混膜CKCS和CKCs的表面性质;模拟体内生理条件研究了葡甘聚糖胶和三元膜CKCS的体外生物降解性，利用体外细胞培养和动态凝血实验、溶血实验等研究了CKCS和CKCs膜的生物相容性;并通过动物实验考察了膜材料的部分医用性能。 4.对C一KGM膜的结构表征和生物学性能研究表明:班值蕊7时，胶原蛋白与葡甘聚糖之间可以任意比例互溶。该条件下共混膜中胶原蛋白与葡甘聚糖之间有较强的相互作用，良好的相容性。经戊二醛交联的胶原蛋白一葡甘聚糖冻干膜具有止血材料所需的多孔结构。C.KGM膜比胶原膜具有更优良的力学性能和更好的热稳定性，但吸水性过强。动物实验表明:交联的冻干膜比明胶海绵具有更好的止血作用，更好的粘附性，一定质量比的胶原蛋白一葡甘聚糖交联冻干膜可达到纯胶原蛋白海绵相近的止血效果，作为一种良好的止血材料，可望在临床上得到广泛应用。 5.三元共混膜CKCS和CKCs的结构表征和生物学性能研究 (1)结构表征和物理性能测试。结果表明:两种三元膜cKcs和cKcs中葡甘聚糖与胶原蛋白和/壳聚糖或软骨素均具有良好的相容性，膜中3种天然高分子之间存在着静电引力、氢键等强烈的相互作用。CKCS膜和CKCs膜均具有均匀而光滑的截面形貌，冻干的CKCS膜具有适宜细胞生长和增殖的多孔结构。N副OH溶液处理使CKCS膜的吸水性等物理性能和生物学性能得到了显著改善。三元膜CKCS力学性能明显优于二元共混膜胶原蛋白一壳聚糖(C一s)及C~KGM和3种单一聚合物膜，其吸水率较C.KGM、胶原膜和葡甘聚糖膜显著降低了，这更有利于生物医学应用:CKCs膜的力学性能和吸水率与二元膜C一KGM和胶原蛋白一软骨素共混膜(c一Cs)，及3种单一聚合物膜比较均有明显改善，但C KCS膜的湿态力学性能较CKCS膜差，吸水性较CKCS膜略强。CKCS膜和CKCs膜均同时具有生物医用材料必备的透水汽性、吸附性和渗透性。DSC和TGA分析均显示:几种膜的热稳定性为CKCS>C~KGM>C〔S四川大学博卜学位论文>壳聚糖(CS)>KGM>CKCs>胶原。 (2)表面性质。郑S对胶原含量分别为40%和50%，KGM与CS质量比不同的CKCS一5膜和CKCS一2膜和含软骨素的胶原含量为65%的CKCs一1膜表面的定性、定址分析表明:与胶原膜比较，3种共混膜表面的CIS、N!s和015的结合能及、}铀圣宽均发生了改变，CKCS一5和CKCS一2膜表面的C一NHZ，C一OH峰在两种膜表面Cls谱中所占比例明显增加，CKCs-1膜表面的C=O和CONHZ峰在共混膜的C!、谱中所占比例大大增加，为最高比例峰。3种膜表面碳含量比胶原膜均明显增加了，而氧、氮含量却明显降低了，C KCs一1膜表面还出现了少量硫元素。表面元素含量的这种变化表明CKCS一5、CKCS一2膜和CKCs一1膜表面极性均较胶原膜降低了，而疏水性相应增高了，因此CKCS膜和CKCs膜表面与极性和亲/疏水性相关的吸水性较胶原膜有显著改善，其与极性和亲/疏水性相关的细胞粘附性和生物相容性等也将随之改变，使用效果较未改性胶原膜将有明显差异。CKCS一5和CKCS一2膜表面xPS很相似，碳更多还原

【Abstract】 Hideoffals as a major part of tannery waste contain over 80% collagen. Collagen is a kind of excellent functional biomaterial as well as a good source of amino acids. Because of its special biological properties, collagen has good application foreground in medicine, food, cosmetic and agriculture.Based on present application status of waste products in curriery in China, the advantages and disadvantages of collagen and analysis and characterization of collagen extracted from hideoffals, studies on collagen application properties and its blend films with one or two polysaccharides were done in this thesis.The contents and results of this thesis are as follows:First, relative molecular weights of collagen and its polypeptides were determined. It is difficult to determine molecular weights of collagen and its polypeptides with relative molecular weights less than 20 kD. Aimed at this, a new method to determine relative molecular weights of small molecular polypeptides was set up. This method is especially suitable for the measurement of relative molecular weights of small molecular collagen and its polypeptides.Second, a systemic analysis on physicochemical properties of collagen and its polypeptides with different relative molecular weights shows that collagen and its polypeptides have good application properties. Determination on viscosity property of collagen and its polypeptides with different relative molecular weights indicates that the viscosity of collagen and its polypeptide solutions is not only changed withtheir relative molecular weights but also can be influenced by various factors such as concentration, temperature, acidity, electrolytes added and keep time.Third, when collagen is used in tissue engineering, it has some drawbacks such as poor mechanical property, higher water absorbability, much lower ability to be moulded at watery condition which is unsuitable for construction of inner organs, much low stability to collagenase produced in the body and its cambium and low ability to synchronize with the growth rate of tissue cells. Based on the above, three novel kinds of collagen-polysaccharide blend films are first made including collagen-Konjac glucomannan blend film (C-KGM), collagen-Konjac glucomannan-chitosan blend film (CKCS) and collagen-Konjac glucomannan chondroitin sulfate blend film film (CKCs). FT infra red analysis (FT-IR) and X-ray diffraction (X-RD) were applied to study morphological structure, mechanical property, water absorption and heat stability of the blend films and the interactions and compatibility of the blend. X-ray photoelectron spectrum (XPS) and determination of surface energy were also used .to study the surface property of CKCS and CKCs. Biodegradability of Konjac glucomannan gel and CKCS in vitro was tested by mimicking physiological conditions in vivo. Biocompatibility of CKCS and CKCs. was determined by cell culture in vitro, dynamic blood coagulation and hemolysis test. In addition, animal experiment was applied to investigate some of medicinal properties of these two blend films.Fourth, results from studies on structure characterization and biological properties results show that collagen can be miscible with Konjac glucomannan at any ratio at pH value of or lower than 7.0 with stronger interactions and good compatibility being present between collagen and Konjac glucomannan in the blend film at this condition. The freeze-dried collagen-Konjac glucomannan film cross linked by glutaraldehyde has a porous structure essential for hemostasis materials. Compared to pure collagen film, the film has higher stability to heat and bettermechanical property. However, this blend film has much stronger water absorbability. Animal experiment shows that cross linked freeze-dried film has better hemostasis and adhesiveness than gelatin sponge, and that a cross linked freeze-dried film prepared by collagen and Konjac glucomannan in a certain ratio can produce hemosatsis close to that of pure collagen sponge: As a good hemostasis material, this film will be更多还原