摘要
目的：分析乌梢蛇Ⅱ型胶原蛋白（Zaocys type Ⅱ collagen，ZC Ⅱ）与其他物种Ⅱ型胶原蛋白（C Ⅱ collagen）的同源性，探讨其对胶原诱导性关节炎（Collagen—induced arthritis，CIA）小鼠关节炎症的影响。方法：采用限制性胃蛋白酶消化法提取ZCⅡ；SDS—PAGE凝胶电泳及紫外分光光度计法对其鉴定，质谱分析其同源性。鸡CⅡ小鼠尾根部皮下注射诱导CIA模型；口服ZCII高、中、低剂量干预，视觉评分法对关节炎评分，HE染色评价小鼠膝关节病理变化。结果：提取的ZCU分子量约为110～140kD，紫外吸收峰为230nm左右；质谱分析所得肽质量指纹图谱与其他物种有4个以上肽段匹配，蛋白评分大于95％。在关节炎症方面，与正常对照组比较，CIA小鼠关节炎及组织病理学评分升高（P〈0．05）；与CIA模型组比较，不同剂量ZCU干预组关节炎及组织病理学评分显著降低（P〈0．05）。结论：质谱分析结果提示ZCⅡ与其他物种CⅡ具有较高同源性；ZCII能显著抑制CIA小鼠的关节肿胀，改善关节组织病理变化。
Objective ： To analyze the homology of Zaocys type Ⅱ collagen （ ZC Ⅱ ） with the C Ⅱ collagen from other species, and to investigate the effect of ZC Ⅱ on arthritis in mice with collagen-induced arthritis（CIA）. Methods ：ZC Ⅱ was purified with restriction pep-sin digestion. Then SDS-PAGE gel electrophoresis and UV spectrophotometry were used to identify the protein, the homology of the ZC II peptide was analyzed with Mass Spectrometry. The model of CIA mice were induced by subcutaneous injection of Chicken C II into male C57BL/6 mice from the base of the tails. After immunization, ZC Ⅱ [ H, M, L ：40,20 and 10 μg/（ kg 丶 d）] was administered orally to mice from day 21 to 28 accordingly. The severity of the arthritis in each limb was evaluated using a macroscopic scoring system, and his- topathological change of joint was observed by light microscope with HE staining. Results ： The molecular weight of ZC Ⅱ protein deter- mined by SDS-PAGE gel electrophoresis was between 110 kD and 140 kD, and UV absorption peak appeared at around 230 nm in wave- length. The peptide mass fingerprinting（PMF） of the purified protein by Mass Spectrometry analysis showed that it had at least 4 pep- tides matched with other species,and the protein score was greater than 95%. Compared with normal group, the CIA model group had significantly higher scores for arthritis and histopathological changes （P 〈 0. 05 ）. Meanwhile, the same scores for the ZC ]] peptide-trea- ted mice with CIA were significantly lower than the mice from CIA model group（ P 〈 0. 05 ）. Conclusion：Results of Mass Spectrometry analysis demonstrate that ZC Ⅱ has high homology with the C Ⅱ from other species. Oral administration of ZC II can suppress arthritis in mice with CIA and ameliorate the histopathological changes of the joint.
出处
《中药材》
CAS
CSCD
北大核心
2014年第6期1020-1024,共5页
Journal of Chinese Medicinal Materials
基金
国家自然科学基金(81173456)
广东省自然科学基金(S2012010009164)
国家教育部博士点基金(20094433110006)
广东省"211"工程三期重点学科建设项目
