本文選題：類風(fēng)濕性關(guān)節(jié)炎 + 組織激肽釋放酶結(jié)合蛋白　； 參考：《華僑大學(xué)》2017年碩士論文

【摘要】：類風(fēng)濕性關(guān)節(jié)炎(rheumatoid arthritis,RA)是主要以關(guān)節(jié)和關(guān)節(jié)附近組織的非感染性炎癥的慢性全身性疾病,該病的主要病理特征是關(guān)節(jié)滑膜的炎癥,炎性細胞浸潤,形成血管翳,軟骨和骨組織破壞,然后導(dǎo)致關(guān)節(jié)畸形和功能喪失,最后可累及漿膜、心、肺及眼結(jié)締組織等多種炎癥性疾病。組織激肽釋放酶結(jié)合蛋白(Kallistatin,Kal)是一種絲氨酸蛋白酶抑制劑,可以與Kallikrien(血管舒緩素)特異性結(jié)合并抑制其功能的發(fā)揮。Kal具有多種生物學(xué)功能如抗炎、抗氧化、抑制新生血管生成、抗腫瘤等。本論文根據(jù)類風(fēng)濕性關(guān)節(jié)炎的病理特點以及組織激肽釋放酶結(jié)合蛋白的生物學(xué)功能,主要以組織激肽釋放酶結(jié)合蛋白為研究對象,從體外細胞實驗及體內(nèi)動物實驗兩方面,考察其對類風(fēng)濕性關(guān)節(jié)炎的作用,并初步探討其抗類風(fēng)濕性關(guān)節(jié)炎的作用機制。本課題的主要研究內(nèi)容包括以下四個方面:1.采用畢赤酵母表達系統(tǒng),高密度發(fā)酵,再經(jīng)過離心、鹽析、疏水層析、超濾濃縮等過程進行純化,然后采用Western blot鑒定Kal蛋白,ELISA測定純化后Kal的濃度,為后續(xù)實驗做好準備。2.采用Ⅱ型膠原酶消化法,分離原代SD大鼠軟骨細胞,通過IL-1β誘導(dǎo)建立軟骨細胞炎癥模型。3.Kal對軟骨細胞炎癥模型的作用,分別通過檢測(1)Kal對軟骨細胞活力的影響;(2)Kal對炎癥軟骨細胞釋放NO的影響;(3)Kal對炎癥軟骨細胞分泌TNF-α的影響;(4)Kal對炎癥軟骨細胞凋亡的影響,來考察Kal對軟骨細胞炎癥模型的作用。結(jié)果顯示,Kal對IL-1β誘導(dǎo)的軟骨細胞炎癥模型,可有效保護由IL-1β引起的細胞損傷,主要表現(xiàn)在:能夠提高軟骨細胞的活力;降低炎癥介質(zhì)、炎癥因子的表達;通過下調(diào)凋亡蛋白Bad的表達,上調(diào)抗凋亡蛋白Bcl-xL的表達,抑制軟骨細胞的凋亡。4.考察Kal對大鼠佐劑性類風(fēng)濕性關(guān)節(jié)炎模型的效果,結(jié)果顯示,在體內(nèi)類風(fēng)濕性關(guān)節(jié)炎動物模型中,Kal能夠有效抑制完全弗氏佐劑引起的炎癥反應(yīng),對大鼠佐劑性類風(fēng)濕性關(guān)節(jié)炎有一定的治療作用,主要表現(xiàn)在:Kal可以降低CFA引起關(guān)節(jié)腫脹,降低炎癥介質(zhì)、炎癥因子的表達。通過體外細胞實驗及體內(nèi)動物實驗,表明Kal通過降低炎癥介質(zhì)、炎癥因子的分泌,下調(diào)凋亡蛋白Bad的表達,上調(diào)抗凋亡蛋白Bcl-xL的表達,抑制軟骨細胞的凋亡,對類風(fēng)濕性關(guān)節(jié)炎起到治療作用。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，

本文編號：2068983