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本实验以猪鼻骨为原料,采用盐析法提取天然Ⅱ型胶原蛋白,采用碱性蛋白酶酶解制备胶原肽,并测定胶原蛋白的纯度,分析胶原蛋白及胶原肽的理化性质。通过构建小鼠CIA动物模型,研究Ⅱ型胶原蛋白及胶原肽对类风湿性关节炎的作用效果。结果表明,提取所得蛋白的胶原蛋白含量为82.86%,在紫外波长230 nm下有最大吸收峰,280 nm处几乎无吸收峰,分子量大于300 KD,具备天然Ⅱ型胶原蛋白特征;酶解得胶原肽不含Ⅱ型胶原蛋白,分子量小于20 KD;Ⅱ型胶原蛋白能够降低小鼠关节指数、降低关节变形率,明显降低小鼠血清中IL-17、MMP-3和TNF-α水平(p<0.05),并能显著提高T细胞刺激指数(P<0.05),对B细胞刺激指数无明显效果(p>0.05);组织病理学显示Ⅱ型胶原蛋白能够减轻关节破坏程度,抑制软骨退化;而胶原肽不具有以上作用。
In this experiment, pig nasal bone was used as raw material, natural type II collagen was extracted by salting-out method, collagenase was prepared by alkaline protease digestion, and the purity of collagen was measured. The physicochemical properties of collagen and collagen peptide were analyzed. The effect of collagen type Ⅱ and collagen peptide on rheumatoid arthritis was studied by constructing mouse CIA animal model. The results showed that the extracted protein had a collagen content of 82.86%, a maximum absorption at 230 nm and a peak at 280 nm with a molecular weight of more than 300 KD. Collagen type II did not contain type II collagen and its molecular weight was less than 20 KD. Type II collagen could reduce joint index, decrease joint deformity, and significantly reduce the levels of IL-17, MMP-3 and TNF-α in serum (p < 0.05), and significantly increased T cell stimulation index (P <0.05), but no significant effect on B cell stimulation index (p> 0.05). Histopathology showed that type Ⅱ collagen could reduce the degree of joint destruction and inhibit cartilage degeneration; Collagen peptide does not have the above effect.