大鼠Ⅱ型胶原蛋白的提取及关节炎动物模型的诱导

Isolation of rat collagen typeⅡto induce collagen-induced arthritis in rats

目的拟建立一种简单易行且经济的大鼠软骨Ⅱ型胶原(collagen typeⅡ,CⅡ)提取方法,并以此构建CⅡ诱导的关节炎动物模型(collagenⅡ-induced arthritis,CIA)。方法将大鼠软骨通过机械法破碎,再结合盐酸胍除去蛋白多糖,胃蛋白酶消化,0.9 mol/L氯化钠盐析,透析冻干等步骤,最终获得纯化CⅡ。经SDS-PAGE电泳鉴定后,诱导大鼠CIA模型。结果SDS-PAGE结果显示可见约130 ku明显条带,无杂带。使用该CⅡ可成功诱导DA大鼠CIA模型,造模成功率100%,在CIA大鼠血浆中可检测到显著升高的抗CⅡ抗体。结论使用本方法可获得纯度较高的CⅡ,可用于CIA模型构建,为类风湿性关节炎的研究提供研究工具。

Objective To establish a simple and economical method to isolate collagen typeⅡ(CⅡ)protein from rat cartilages and to induce collagen-induced arthritis(CIA)by using purified CⅡ.Methods Rat cartilages were collected and homogenated mechanically.Guanidine buffer was used to remove proteoglycans in cartilages,and other components were digested by pepsin.CⅡwas salted out by 0.9 mol/L sodium chloride,dialyzed and lyophilized finally.The isolated CⅡwas identified by SDS-PAGE and was used to induce CIA model in DA rats.Results Results of SDS-PAGE showed a clear band around 130 ku in the gel,and that there was no non-specific band.CIA model was successfully induced in DA rats by using CⅡthat we had obtained,with a 100%prevalence rate.Most importantly,anti-CⅡantibody level in the plasma of CIA rats was significantly increased.Conclusion CⅡwith high purity could be isolated by using our method,and further be used to induce CIA model which provides a useful tool for research on rheumatoid arthritis.