【作者】 曹慧；

【导师】 许时婴；

【作者基本信息】 江南大学 ， 食品科学， 2008， 博士

【摘要】 鸡骨是肉鸡加工的主要副产物,目前大量鸡骨没有得到充分的开发和利用,而是当作废弃物抛弃,造成极大的浪费,且污染环境。Ⅱ型胶原是鸡胸软骨中的主要成分,Ⅱ型胶原独特的结构和功能使其广泛应用于食品、医药及化工等领域。从鸡胸软骨中提取Ⅱ型胶原,不但充分利用了鸡骨资源,减少环境污染,解决了家禽加工资源综合利用的问题,同时也可以得到一种具有高附加值的生物活性物质,具有很大的理论意义和实用价值。本课题采用酶法从鸡胸软骨中制备Ⅱ型胶原,并对其生物活性和结构进行了研究,主要研究内容如下:采用高效液相色谱法建立了Ⅱ型胶原的测定方法。色谱条件为:流动相,A为5%乙腈、0.05%TFA,B为80%乙腈(v/v);采用线形梯度洗脱;检测波长,220 nm;流速,1 mL/min。柱温,35℃,进样量,10μL。高效液相色谱法对Ⅱ型胶原进行定量检测与传统的WoessnerⅠ法具有良好的相关性(R2>0.99)。高效液相色谱法在保证准确性和重现性的基础上,具有省去繁琐耗时的样品预处理、所需样品用量少、易于自动化的优点,从而为Ⅱ型胶原提供了一种新的测定方法。确定了从鸡胸软骨中制备Ⅱ型胶原的工艺路线。首先采用甲醇:氯仿(2:1)低温浸泡除去脂肪,使脂肪的残留率降至0.072%;再用1 mol/L的NaCl溶液浸泡骨料并缓慢搅拌除去大部分盐溶性的杂蛋白;采用胃蛋白酶切除Ⅱ型胶原的端肽,使Ⅱ型胶原由不溶性转为可溶性,胃蛋白酶解温度、时间及胃蛋白酶浓度显著影响Ⅱ型胶原的提取率及二级结构。胃蛋白酶提取鸡胸软骨Ⅱ型胶原的最佳工艺条件为:胃蛋白酶浓度为1%(w/v),酶解温度20℃,酶解时间36h,提取率为43.49%,Ⅱ型胶原保持着比较完整的三股螺旋结构。采用NaCl沉淀酶解液中的Ⅱ型胶原,Ⅱ型胶原最佳的盐析工艺条件为:氯化钠浓度为3 mol/L,盐析温度20℃,盐析时间为24h,回收率为85.93%。采用DEAE-sepharose CL 6B离子交换色谱分离Ⅱ型胶原与糖胺聚糖,由SDS - PAGE及RP-HPLC测定结果表明:鸡胸软骨Ⅱ型胶原样品与Sigma公司标准品及关节软骨Ⅱ型胶原样品的电泳谱带相同,具有较高的α带和少量的β二聚体。图谱上无其他杂带,经纯化后的Ⅱ型胶原的纯度较高。研究了Ⅱ型胶原的结构及理化性质。结果表明Ⅱ型胶原中甘氨酸、羟脯氨酸和丙氨酸含量最高,每1000个氨基酸残基中分别为310、117及115。组氨酸和酪氨酸含量较低,没有检测出色氨酸,为典型动物胶原的氨基酸组成。园二色性及红外测定结果表明Ⅱ型胶原是由三条相同的α链构成的螺旋。Ⅱ型胶原的变性温度为43.843℃,在220 nm附近产生最大的光吸收,在pH值为5.8时,溶解度达到最低。由AFM(原子力显微镜)测定结果表明,Ⅱ型胶原原纤维由一个个成念珠状的结构紧密连接而成,每个念珠即为一个横纹周期(D周期),平均每个周期为65 nm,每个胶原分子的长度约为300 nm,直径约为1.5 nm。Ⅱ型胶原的周期结构对应明带和暗带构成的高低起伏的波状结构,明带对应波峰,暗带对应波谷,每个周期的波峰和波谷有5 nm左右的高度差。以EDC/NHS为交联剂制备Ⅱ型胶原-硫酸软骨素支架,并对Ⅱ型胶原-硫酸软骨素支架修复软骨损伤的功能进行研究。结果表明,EDC/NHS交联增加了胶原支架的热稳定性及对胶原酶的抗性,并降低了游离氨基酸的含量及含水率,从而改善了胶原分子作为细胞支架易降解的缺点。最适的EDC的添加浓度为7 mg/mL。Ⅱ型胶原与硫酸软骨素复合后,细胞亲和性大大提高,软骨细胞能很好的在复合支架上粘附、生长和增殖,并保持软骨细胞特异分化的表型,分泌Ⅱ型胶原与蛋白多糖。培养14天后已有软骨样组织形成,这表明Ⅱ型胶原-硫酸软骨素支架适合作为软骨组织工程研究的细胞载体材料。通过建立大鼠类风湿性关节炎模型,研究了Ⅱ型胶原对人类类风湿性关节炎的防治作用。采用Ⅱ型胶原诱导的大鼠类风湿性关节炎症状主要表现为关节软骨破坏,增生性滑膜炎,并伴有单个核细胞浸润并持续存在于滑膜中。从测得的与RA紧密联系的三种炎性细胞因子水平来看,口服Ⅱ型胶原和氨基葡萄糖显著抑制了RA的发展,提示其治疗作用机理可能与抑制滑膜细胞分泌炎性细胞因子,纠正Th1和Th2细胞平衡失调有关,也进一步表明了Ⅱ型胶原和硫酸氨基葡萄糖作为防治类风湿性关节炎保健品的可行性。更多还原

【Abstract】 Chick bone is the main by-products of chick manufacturing industry, most of chick bone have not been exploited and are directly discharged into estuaries resulting in environmental pollution. Type II collagen is the main structural component of chick sternal cartilage and is used in various food applications, pharmaceutical applications and chemical industrial with specific molecular structure. If the type II collagen can be extracted from the chick sternal cartilage, not only the polluting problem can be resolved, but also can obtain a kind of biological activity product with high appending value.Pepsin was used to extract type II collagen from chick sternal cartilage and the biological activities and the structure of type II collagen were studied in this paper. Major content of this paper as following:Reversed-phase high performance liquid chromatography (RP-HPLC) method was demonstrated to determine the content of type II collagen. Chromatography was performed with an injection volume of 10μl. The mobile phase consisted of two solvent: (A) 5% acetonitrile and 0.05% trifluoroacetic (TFA) and (B) 80% acetonitrile (v/v). The separation was performed using the linear gradient of A-B (v/v). Flow rate was maintained 1 ml/min. Absorbance was monitored at 220 nm. The RP-HPLC method had a good linearity correlation with Woessner I method (R2>0.99). As compared with the conventional method, the proposed method has proved to be convenient, sensitive, accurate and reproducible on base of avoiding the use of a time-consuming pretreatment procedure.Technical route of extracting type II collagen from chick sternal cartilage were confirmed. The chick sternal cartilage was defatted with chloroform-methanol (2:1, v/v) and the residual fat was 0.072%. Then, the sternal cartilage was extracted using 1.0 mol/L NaCl in 0.05 mol/L Tris-HCl (pH 7.5) at 4°C for 24 h to remove contaminating protein. After the extracts were aggregated by centrifugation, the digestion was tested by mixing precipitation with pepsin to assess the temperature, times and the ratio of enzyme to precipitation effect on the extracting ratio and secondary structure of type II collagen. The optimum technical parameters for pepsin digestion were studied: extraction times for 32h, pepsin concentration for 0.5% and temperature for 20°C. It seemed, therefore, the pepsin-solubilized type II collagen of higher stability and extracting ratio can be obtained (43.49%).NaCl was applied to precipitate type II collagen. The optimum technical parameters for NaCl salting out were studied: extraction times for 32h, pepsin concentration for 0.5%, temperature for 20°C and the recovery ratio was 85.93%.DEAE-sepharose CL 6B chromatography provided a rapid and efficient method for separating collagen from proteoglycans in cartilage extracts. The results of SDS-PAGE and RP-HPLC suggested the prepared type II collagen essentially was free from contaminating proteins by the method used in this study for purification. All these collagen samples purified had similar migration bands and consisted ofαchain and their dimers (βchains) with a subunit Mr of 110 kDaThe structure and composition of type II collagen were studied. Type II collagen had a high content of glycine, hydroxyproline and proline residues, with 310, 117 and 115 residues per 1000 amino acids residues respectively, and small amounts of tyrosine, cysteine, histidine and methionine residues with 5, 18 and 10 residues per 1000 amino acids residues respectively. The Td was determined to be about 42°C for purified type II collagen and about 44°C for intact cartilage. The minimal difference between denaturation temperature of purified type II collagen and intact cartilage indicate that purification process has little influence for stability of collagen.Type II collagen molecules consisted of three polypeptide chains, which self-assemble into D-periodic cross-striated fibrils with 65 nm. Collagen molecules, forming the fibril, consisted of an uninterrupted right handed triple helix called tropocollagen, approximately 300 nm in length and 1.5 nm in diameter. The stagger of molecules gives rise to a characteristic band pattern of light and dark regions and viewed using an electron microscope.The porous type II collagen-chondroitin sulfate (CS) scaffold were prepared using variable concentrations of 1-ethyl-3(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). The results suggested that EDC - crosslinking increased the denaturation temperature (Td) and resistance to enzymatic degradation by bacterial collagenase. A significant decrease in the swelling ratio and free amino groups per 1000 amino acid residues was also detected. The mechanical stability of collagen-CS scaffolds were improved with EDC concentration for 7 mg/ml. Then, isolated chondrocytes were cultured in porous type II collagen scaffolds either in the presence and/or absence of covalently attached CS up to 14 days. Cell proliferation and the total amount of proteoglycans and type II collagen retained in the scaffolds were higher in type II collagen-CS scaffolds. Histological analysis showed the formation of a denser cartilaginous layer at the scaffold periphery. Scanning electron microscopy revealed chondrocytes distributed the porous surface of both scaffolds maintained their spherical morphology. The results of the present study also indicate that type II collagen-CS scaffolds have potential for use in tissue engineering.The effect of type II collagen (CII) prepared from the chick sternal cartilage and Glucosamine Sulfate (GLS) on collagen-induced rat rheumatoid arthritis (RA) model (CIA) was studied. CIA was established in male Sprague-Dawley (SD) rats with intradermal injection of chicken type II collagen at the left hind paw of the animals, and then CIA rats were treated daily using oral administration of different doses of CII with or without GLS beginning on the day of the induction of arthritis (day 0, the prophylactic treatment) until day 45. Histopathologic sections of diarthrodial joints showed that injection of type II collagen with CFA produced a chronic proliferative synovitis which secondarily destroyed articular cartilage and bone. Synovium of the onset of arthritis showed marked edema and infiltration by dense aggregates of mononuclear cells and occasional neutrophils. Prophylactic treatment with type II collagen significantly suppressed the onset of arthritis and markedly reduced paw swelling even in the established CIA. The therapy may be achieved by restraining cytokines excreted by synoviocyte and balance the Th1 and Th2 cell. Hence, our studies demonstrate the quality, safety, and effectiveness of type II collagen as an anti-arthritic agent, which makes type II collagen a strong candidate for further clinical trials on rheumatoid arthritis (RA) patients.更多还原