【摘要】 目的提取纯化Ⅱ型胶原蛋白(CⅡ)并制备胶原诱导性关节炎(CIA)大鼠模型。方法采用胃蛋白酶消化法从鸡胸骨剑突上提取CⅡ,通过DEAE-52纤维树脂处理、NaC l的反复盐析纯化并采用SDS-PAGE鉴定和吸收光谱分析。所提CⅡ加以完全弗氏佐剂采用皮内注射的方法建立大鼠C IA模型。容积法检测大鼠足爪肿胀度并进行评分;HE染色法对关节组织作病理检查;ELISA法检测体内抗CⅡ抗体的含量;放免法检测血清细胞因子IL-1β、TNFα-含量。结果自提的CⅡ与标准品分子量一致,最大吸收峰为226 nm。CIA大鼠足爪的炎症反应强烈,关节肿胀明显;病理检查关节组织可见其组织纤维化,伴有大量炎性细胞浸润;血清中抗CⅡ抗体水平和IL-1β、TNFα-显著升高。结论该方法提纯CⅡ简单、高效,用以制备大鼠CIA模型成功。更多还原

【Abstract】 Objective To isolate and purify collagen type Ⅱ(CⅡ) and establish the rat model of collagen-induced arthritis(CIA).Methods CⅡ was extracted from chicken sternal cartilage by pepsin digestion,and purified through treatment with DE52 cellulose,salting of sodium chloride,then identified by SDS-PAGE analysis and absorption spectrum.The rats were injected subcutaneously with CⅡ,emulsified with complete Freund′s adjuvant(CFA).The change of secondary paw-swelling was observed and scored;Pathological changes of synovium were examined by Hematoxylineosin(HE)stain method;antibody against CⅡ was determined by enzyme-linked immunosorbent assay(ELISA);Interleukin-1(IL-1)and Tumor necrosis factor alpha(TNF-α) were measured by Radioimmunoassay.Results We found that the molecular size of purified CⅡ was near to standard CⅡby SDS-PAGE and the absorption peak was at 226 nm.The inflammation and paw swelling of CIA rats increased significantly;The pathological examination showed that pannus formed and inflammatory cells infiltrated into the synovium;The serum antibodies against CⅡ,IL-1 and TNF-α were elevated significantly.Conclusion The method of preparing CⅡ is simple and effective,CIA rat model could be successfully established by subcutaneous injection of CⅡ emulsified with CFA.更多还原