组织激肽释放酶结合蛋白对类风湿性关节炎的作用及其机制探究

发布时间：2018-06-26 04:00

  本文选题：类风湿性关节炎 + 组织激肽释放酶结合蛋白　； 参考：《华侨大学》2017年硕士论文

【摘要】：类风湿性关节炎(rheumatoid arthritis,RA)是主要以关节和关节附近组织的非感染性炎症的慢性全身性疾病,该病的主要病理特征是关节滑膜的炎症,炎性细胞浸润,形成血管翳,软骨和骨组织破坏,然后导致关节畸形和功能丧失,最后可累及浆膜、心、肺及眼结缔组织等多种炎症性疾病。组织激肽释放酶结合蛋白(Kallistatin,Kal)是一种丝氨酸蛋白酶抑制剂,可以与Kallikrien(血管舒缓素)特异性结合并抑制其功能的发挥。Kal具有多种生物学功能如抗炎、抗氧化、抑制新生血管生成、抗肿瘤等。本论文根据类风湿性关节炎的病理特点以及组织激肽释放酶结合蛋白的生物学功能,主要以组织激肽释放酶结合蛋白为研究对象,从体外细胞实验及体内动物实验两方面,考察其对类风湿性关节炎的作用,并初步探讨其抗类风湿性关节炎的作用机制。本课题的主要研究内容包括以下四个方面:1.采用毕赤酵母表达系统,高密度发酵,再经过离心、盐析、疏水层析、超滤浓缩等过程进行纯化,然后采用Western blot鉴定Kal蛋白,ELISA测定纯化后Kal的浓度,为后续实验做好准备。2.采用Ⅱ型胶原酶消化法,分离原代SD大鼠软骨细胞,通过IL-1β诱导建立软骨细胞炎症模型。3.Kal对软骨细胞炎症模型的作用,分别通过检测(1)Kal对软骨细胞活力的影响;(2)Kal对炎症软骨细胞释放NO的影响;(3)Kal对炎症软骨细胞分泌TNF-α的影响;(4)Kal对炎症软骨细胞凋亡的影响,来考察Kal对软骨细胞炎症模型的作用。结果显示,Kal对IL-1β诱导的软骨细胞炎症模型,可有效保护由IL-1β引起的细胞损伤,主要表现在:能够提高软骨细胞的活力;降低炎症介质、炎症因子的表达;通过下调凋亡蛋白Bad的表达,上调抗凋亡蛋白Bcl-xL的表达,抑制软骨细胞的凋亡。4.考察Kal对大鼠佐剂性类风湿性关节炎模型的效果,结果显示,在体内类风湿性关节炎动物模型中,Kal能够有效抑制完全弗氏佐剂引起的炎症反应,对大鼠佐剂性类风湿性关节炎有一定的治疗作用,主要表现在:Kal可以降低CFA引起关节肿胀,降低炎症介质、炎症因子的表达。通过体外细胞实验及体内动物实验,表明Kal通过降低炎症介质、炎症因子的分泌,下调凋亡蛋白Bad的表达,上调抗凋亡蛋白Bcl-xL的表达,抑制软骨细胞的凋亡,对类风湿性关节炎起到治疗作用。表明Kal具有良好的抗RA作用。
[Abstract]:Rheumatoid arthritis (RA) is a chronic systemic disease characterized by inflammation of synovial membrane, infiltration of inflammatory cells, and formation of pannus. Cartilage and bone tissue destruction, then leading to joint deformities and loss of function, finally involving serosa, heart, lung and eye connective tissue and other inflammatory diseases. Tissue Kallistate releasing enzyme binding protein (Kal) is a serine protease inhibitor, which can specifically bind to Kallikrien and inhibit its function. Kal has many biological functions such as anti-inflammation, anti-oxidation, and inhibiting angiogenesis. Antitumor, etc. According to the pathological characteristics of rheumatoid arthritis and the biological function of tissue kallikrein binding protein, the tissue kallikrein binding protein was studied in vitro and in vivo animal experiments. To investigate its effect on rheumatoid arthritis and the mechanism of its anti-rheumatoid arthritis. The main research contents of this subject include the following four aspects: 1. Using Pichia pastoris expression system, high density fermentation, then centrifugation, salting out, hydrophobic chromatography, ultrafiltration concentration and other processes for purification, then Western blot identification of the concentration of purified Kal protein Elisa for the preparation of the follow-up experiment. 2. Primary SD rat chondrocytes were isolated by type 鈪，

本文编号：2068983