桂枝芍药知母汤对Ⅱ型胶原诱导的关节炎大鼠作用机制的实验研究

【作者】 赵慧；

【导师】 顾立刚；

【作者基本信息】 北京中医药大学 ， 中西医结合基础， 2006， 硕士

【摘要】 研究目的:类风湿性关节炎(rheumatoidarthritis,RA)是一种慢性进行性的自身免疫性疾病,以关节滑膜炎及对称性、破坏性的关节病变为主要特征。RA的发病率在我国约为0.4%,全球为0.5%～1.0%。半个世纪以来,关于RA的发病机制一直处在不断的发展变化中。其中滑膜细胞、软骨细胞、巨噬细胞等产生的细胞因子和滑膜细胞的类肿瘤样增生在RA免疫炎症和骨质破坏中起着重要的作用,是研究热点。中药复方桂枝芍药知母汤是金匮要略的名方,其方祛风除湿、温经散寒、滋阴清热,临床广泛使用,治疗类风湿关节炎效果显著,但其治疗机制尚不清楚。Ⅱ型胶原诱导性大鼠关节炎模型(collagen-induced arthritis CIA)模型在免疫病理、临床表现、组织病理变化等方面与人RA最为接近,是目前国际公认的研究RA发病机理、治疗药物的理想动物模型。在本实验中,我们复制了Ⅱ型胶原诱导性大鼠关节炎模型,然后从桂枝芍药知母汤对CIA大鼠的治疗作用,血清细胞因子的影响,巨噬细胞功能的影响,对CIA大鼠c-myc mRNA表达影响等方面对其机制进行研究,为临床用药提供理论依据。实验方法:本实验主要分为三个部分,以CIA大鼠作为动物模型,探讨桂枝芍药知母汤治疗RA的作用机制。第一部分建立CIA大鼠模型。实验动物随机分为三组,即正常对照组,CIA模型组和佐剂对照组。其中CIA模型组以含Ⅱ型胶原的完全弗氏佐剂免疫,致炎后第14天发生继发性肿胀,即右侧足肿胀。观察记录大鼠的一般状况,不同时间点各组大鼠继发侧关节的肿胀度的变化,并绘制变化曲线。在免疫42天后,断头处死大鼠,取血清,用ELISA法测定CIA大鼠血清Ⅱ型胶原抗体的含量。第二部分实验,Wistar大鼠CIA致敏第18天后,桂枝芍药知母汤大、中剂量(37.2、18.6g/kg,分别相当于成人每日常用量的20、10倍),灌胃给药。雷公藤多甙片研成细末,加生理盐水,配成混浊液10g/kg.d,连续给药3周。正常组、CIA模型组均灌以等容积蒸馏水。给药三周后,大鼠断头处死。用Griess法测定大鼠血清中NO的水平;用胸腺细胞法测定大鼠血清IL-1的含量。用ELISA法检测TNF-α、IL-10含量的变化。用Griess法测定大鼠腹腔巨噬细胞NO产生能力;中性红比色法测定巨噬细胞的吞噬功能,胸腺细胞法测定巨噬细胞产生IL-1的能力。用ELISA法检测巨噬细胞分泌的TNF-α含量。通过以上指标的测定,来探讨桂枝芍药知母汤对RA的治疗机理。第三部分实验用RT-PCR的方法检测CIA大鼠滑膜细胞c-myc mRNA的表达,从原癌基因的过度表达引起滑膜增生角度来探讨桂枝芍药知母汤对RA的治疗作用。实验结果:第一部分实验发现,致炎后14天大鼠开始出现关节红肿,首先出现红肿的是右后足趾关节,然后蔓延到前足和尾部,并且日趋严重。关节肿胀于第21天达到高峰。发病过程伴随大鼠体温升高和体重减轻。随着时间的延续肿胀逐渐消退,但更多还原

【Abstract】 OBJECTIVE: Rheumatoid arthritis (RA), a common and severe disease, is a chronic inflammatory disease of unknown cause, which is characterized by synovial hyperplasia, inflammation, and joint destruction. Its prevalence in adults in China is about 0.4%, which is lower than that in otber places 1% . And it is one of major diseases causing disability in our nation.The major pathological change of RA is synovitis, with the invasion of cartilage and joint. Among so many factors causing RA, cytokines secreted by synoviocyte,macrophage and the malignant proliferation of synoviocyte are critical to the occurence and development of RA.The TypeⅡcollagen-induced arthritis(CIA) is very similar with RA in many ways of clinical symptoms, immuno pathological and histopathological changes.CIA is a best animal model that is used to study RA. Guizhishaoyaozhimu(GZSY) decotion is one of the most effective medicines for RA, which has been applied in clinics for years ,but the mechanism of GZSY against RA is not clear. In our studies we plan to explore such mechanism of GZSY in RA by using the animal model of CIA rats: the effects of cure of GZSY on CIA rats; the effects of GZSY on CKs in the serum of CIA rats; the effects of GZSY on the function of macrophage from rats; and the influence of GZSY on the mRNA expression of c-myc in synoviocyte of CIA rats.METHODS: By using the pathological models of CIA, we studied the mechanism of GZSY in RA disease. There are totally three parts in my thesis. In the first part,the CIA rats was successful established by injection the mixture of TypeⅡcollagen with Fraud’s adjuvants. During this period, the changes of the general status, swelling of the rats have been recorded on different time. After 42 days of immunization, the rats were put to death and the serum was collected. And then in the following experiments, the antibody of TypeⅡcollagen in the serum of CIA rats has been detected by ELISA.And pathomorphology by HE staining. In the second part, the CIA rats was feed by GZSY 37.2 g/kg and18.6g/kg respectively every day for three weeks. During this period, the changes of the general the changes of the general status, swelling of the rats have been recorded on different time. After 21 days of treatment, the rats were put to death and the serum was collected.The level of TNF-α、IL-10 in the serum was measured by ELISA method, the activity of IL-1 in blood serum was investigated by thymocyte cell proliferation,and nitrogen monoxide yield competence by Griess assay; peritoneal macrophage phagofunction was detected by neutral red assay, nitrogen monoxide yield competence by Griess assay ,the level of TNF-αsecreted by peritoneal macrophage was measured by ELISA method, the activity of IL-1 secreted by peritoneal macrophage was investigated by thymocyte cell proliferation.In the third part ,we investigated the mRNA更多还原