【摘要】：類風(fēng)濕性關(guān)節(jié)炎(rheumatoid arthritis,RA)是一種以關(guān)節(jié)滑膜炎癥為特征的自身免疫性疾病,致殘性強(qiáng),可導(dǎo)致不可修復(fù)的關(guān)節(jié)畸形、破壞及功能喪失。腫瘤壞死因子-α(TNF-α,Tumor Necrosis Factor-alpha)在RA病變的滑膜組織增生、自身免疫反應(yīng)和炎癥等生理病理過程中起著重要作用,處在細(xì)胞因子調(diào)節(jié)網(wǎng)絡(luò)的頂點(diǎn),是RA發(fā)病機(jī)制中處于關(guān)鍵位置的促炎性因子。TNF-α的拮抗劑已被證明是類風(fēng)濕關(guān)節(jié)炎的有效治療劑,但缺點(diǎn)是靶向性低,副作用多。 含額外結(jié)構(gòu)域B的纖維連接蛋白(Extra-domain B of fibronectin, ED-B FN)在RA患者的關(guān)節(jié)炎滑膜中呈現(xiàn)特異性表達(dá)。因此,以ED-B FN單克隆抗體或抗體片段作為靶向載體,與TNF-α單克隆抗體或抗體片段連接,構(gòu)建抗TNF-α抗ED-B FN的雙特異性抗體(Bispecific antibody)αEBTA,在RA的靶向治療中能降低或消除其對正常組織和關(guān)節(jié)的免疫抑制作用,能有效提高藥物的靶向性和安全性。 本研究通過由基因工程方法構(gòu)建的畢赤酵母菌,分泌表達(dá)雙抗aEBTA,上清中雙抗的濃度為3μg／mL,經(jīng)過鎳離子親和層析后,濃度達(dá)到了0.3mg/mL。對其進(jìn)行了體外藥效分析,AIA(佐劑誘導(dǎo)型)模型小鼠藥代動力學(xué)和藥效學(xué)研究。并由結(jié)果分析出雙抗aEBTA能對抗TNF-a的細(xì)胞毒作用；雙抗在類風(fēng)濕關(guān)節(jié)模型(AIA)小鼠炎癥關(guān)節(jié)中呈現(xiàn)特異性聚集,濃度比在其他器官(肝、腎、脾、肺)有顯著差異；藥效學(xué)實(shí)驗(yàn)表明雙抗對AIA小鼠有較好的治療效果。 結(jié)論：雙抗aEBTA對AIA小鼠炎癥關(guān)節(jié)有靶向分布和較好的治療效果,為以后相關(guān)藥物的應(yīng)用和開發(fā)打下了很好的基礎(chǔ)。
[Abstract]:Rheumatoid arthritis (rheumatoid arthritis,RA) is an autoimmune disease characterized by synovial inflammation. It is highly disabled and can lead to irreparable deformities, destruction and loss of function. Tumor necrosis factor-偽 (TNF- 偽, Tumor Necrosis Factor-alpha) plays an important role in the physiological and pathological process of RA disease, such as synovial tissue proliferation, autoimmunity and inflammation, and is at the apex of cytokine regulation network. The antagonist of TNF- 偽 has been proved to be an effective therapeutic agent for rheumatoid arthritis (RA), but its disadvantage is low targeting and many side effects. Fibronectin (Extra-domain B of fibronectin, ED-B FN) containing extra domain B was specifically expressed in synovium of RA patients with arthritis. Therefore, using ED-B FN monoclonal antibody or antibody fragment as target vector, the bispecific antibody (Bispecific antibody) 偽 EBTA, against TNF- 偽 anti ED-B FN was constructed by ligating with (Bispecific antibody) 偽 monoclonal antibody or antibody fragment. The immunosuppressive effect on normal tissues and joints can be reduced or eliminated in the targeted therapy of RA, and the targeting and safety of the drug can be improved effectively. In this study, Pichia pastoris was constructed by genetic engineering method. The concentration of double antibody in the supernatant of double antibody aEBTA, was 3 渭 g / mL, and the concentration was 0.3 mg / mol 路L-1 after Ni ~ (2 +) affinity chromatography. Pharmacodynamics and pharmacodynamics of, AIA (adjuvant-induced) mice were studied in vitro. The results showed that biantibody aEBTA could antagonize the cytotoxic effect of TNF-a, and the biantibody showed specific aggregation in inflammatory joints of rheumatoid joint model (AIA) mice, and the concentration was significantly different compared with other organs (liver, kidney, spleen, lung). Pharmacodynamic experiments showed that the double antibody had a good therapeutic effect on AIA mice. Conclusion: biantibody aEBTA has targeted distribution and good therapeutic effect on inflammatory joints in AIA mice, which lays a good foundation for the application and development of related drugs in the future.
【學(xué)位授予單位】：湖北大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R96

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本文編號：2436027