本文選題：雷公藤紅素 + 聚合物膠束�。� 參考：《上海交通大學》2015年博士論文

【摘要】：研究目的:構建偶聯(lián)抗人FcεRIαFab包裹雷公藤紅素的聚合物膠束(抗人FcεRIαFab-聚合物膠束-雷公藤紅素三聚物),分析其對肥大細胞的靶向性、增殖抑制及促凋亡作用,探討其在過敏性哮喘小鼠體內的分布及對哮喘和被動皮膚過敏反應的抑制效果。研究方法:(1)采用薄膜水化法制備雷公藤紅素-聚合物膠束,EDC偶聯(lián)法構建抗人FcεRIαFab-聚合物膠束-雷公藤紅素三聚物,采用粒度分析儀、透射電子顯微鏡分別觀察膠束的粒徑和形態(tài),反相-高效液相色譜法測定其包封率和載藥量。(2)體外以人外周血嗜堿性白血病細胞KU812(表面高表達FcεRI,靶細胞)和人未成熟肥大細胞HMC-1(表面幾乎不表達FcεRI,非靶細胞)為研究對象,以香豆素6為探針,采用流式細胞儀和激光共聚焦顯微鏡檢測三聚物的靶向作用;CCK-8法測定細胞增殖抑制率,Annexin V-FITC/PI雙標記合并流式細胞儀定量分析凋亡細胞比率,酶法和免疫印跡分別檢測凋亡分子caspase 3活性和切割的PARP表達水平,分析三聚物對肥大細胞增殖抑制及凋亡作用。(3)以卵清蛋白免疫babl/c小鼠建立哮喘模型,以近紅外熒光素dir為探針,觀察三聚物在過敏性哮喘小鼠體內的分布及其對肺組織的靶向性;給予三聚物干預治療,elisa法檢測血清ova特異性ige(ova-sige)和肺泡灌洗液(balf)組胺的水平,采用luminex技術測定balf中th1/th2細胞因子的含量,計數(shù)balf白細胞總數(shù)及嗜酸性粒細胞數(shù),觀察肺組織炎癥情況,分析三聚物對過敏性哮喘的治療效果。此外,通過皮內注射ige-dnp和尾靜脈注射dnp-hsa誘導小鼠被動皮膚過敏反應,給予三聚物干預治療,觀察伊文氏藍擴散程度,驗證三聚物對過敏反應的抑制作用。研究結果:(1)制備了抗人fcεriαfab-聚合物膠束-雷公藤紅素三聚物,包封率為98.9±3.3%,載藥量為22.8±1.0%,粒徑在93.43nm左右,分布均勻,呈短棒狀或橢圓狀。(2)流式細胞儀和激光共聚焦顯微鏡檢測結果顯示抗人fcεriαfab-聚合物膠束進入靶細胞的量顯著高于未偶聯(lián)抗體的膠束;細胞增殖和凋亡的結果表明抗人fcεriαfab-聚合物膠束-雷公藤紅素三聚物作用組的肥大細胞抑制率、凋亡細胞比例、caspase3活性和切割的parp水平均顯著高于單純的雷公藤紅素和雷公藤紅素-聚合物膠束組。(3)體內研究結果表明,三聚物能促進藥物在過敏性哮喘小鼠的肺組織聚集,并有效地減少哮喘小鼠血清ova-sige、balf組胺和th2細胞因子(il-4、il-5、il-13和tnf-α)的合成,增加抗炎因子il-10的分泌,降低balf白細胞和嗜酸性粒細胞數(shù)量,減輕肺組織炎細胞浸潤和黏液分泌;此外三聚物能有效減少伊文氏藍的滲出,降低血管通透性,減輕小鼠被動皮膚過敏反應。且三聚物的抗過敏效果明顯比單純的雷公藤紅素和雷公藤紅素-聚合物膠束組更強。研究結論:成功構建了抗人FcεRIαFab-聚合物膠束-雷公藤紅素三聚物,該三聚物具有良好的表征和肥大細胞靶向性,能特異地抑制肥大細胞增殖、誘導其凋亡;在體內能促進藥物在過敏性哮喘小鼠肺部聚集,對小鼠過敏性哮喘和被動皮膚過敏反應具有明顯的抑制作用。這些結果提示抗人FcεRIαFab-聚合物膠束-雷公藤紅素三聚物具有治療過敏及肥大細胞相關疾病的潛在應用價值。
[Abstract]:Objective: to construct a polymer micelle (anti human Fc e RI alpha Fab- polymer micelle - tripterine trimer) coupled with Fc - epsilon RI - Fab, to analyze its targeting, proliferation inhibition and apoptosis, and to explore its distribution in allergic asthmatic mice and the allergic reaction to asthma and passive skin. The research methods: (1) using the film hydration method to prepare tripterin polymer micelle and EDC coupling method to construct a human Fc epsilon RI Fab- polymer micelle - tripterin trimer. The particle size and morphology of the micelles were observed by the particle size analyzer and transmission electron microscope respectively. The encapsulation efficiency was determined by reversed phase high performance liquid chromatography. (2) (2) the target of human peripheral blood basophilic leukemia cell KU812 (surface high expression Fc epsilon RI, target cells) and human immature mast cells HMC-1 (the surface almost non expression Fc e RI, non target cells) as the research object, with coumarin 6 as the probe, using flow cytometry and laser confocal microscope to detect the target effect of the tripolymer, CCK-8 method The inhibitory rate of cell proliferation was determined. Annexin V-FITC/PI double labeling combined with flow cytometry was used to quantify the apoptotic cell ratio. The activity of caspase 3 and the level of PARP expression were detected by enzyme and immunoblotting respectively. The inhibition and apoptosis effect of tripolymer on mast cell proliferation and apoptosis were analyzed. (3) to establish asthma in babl/c mice immunized with ovalbumin. The model, using the near infrared fluorescein dir as a probe, observed the distribution of the tripolymer in the allergic asthmatic mice and the targeting of the lung tissue. The level of ova specific IgE (ova-sige) and alveolar lavage fluid (BALF) histamine in serum was detected by ELISA, and the content of th1/th2 cytokine in BALF was measured by Luminex technique. Count the total number of BALF white blood cells and eosinophils, observe the inflammation of lung tissue and analyze the therapeutic effect of tripolymer on allergic asthma. In addition, dnp-hsa induced passive skin anaphylaxis in mice by intradermal injection of ige-dnp and caudal vein is induced by intradermal injection of dnp-hsa, and the diffusion degree of Evans blue is observed and the trimer is verified. The research results were as follows: (1) the Fc epsilon RI alpha fab- polymer micelle - tripterin trimer was prepared, the encapsulation rate was 98.9 + 3.3%, the drug loading was 22.8 + 1%, the particle size was around 93.43nm, and the distribution was uniform, and was short rod or ellipse. (2) flow cytometry and laser confocal microscopy showed the anti human Fc e RI alpha Fab - the amount of polymer micelles entered target cells was significantly higher than that of unconjugated micelles. Cell proliferation and apoptosis showed that the mast cell inhibition rate, the percentage of apoptotic cells, the Caspase3 activity and the PARP level of the cleavage group were significantly higher than those of the simple tripterin and the PARP level of the Fc epsilon RI alpha fab- polymer micelle. Tripterine polymer micelle group. (3) in vivo studies have shown that the trimer can promote the accumulation of drugs in the lung tissue of allergic asthma mice and effectively reduce the combination of serum ova-sige, BALF histamine and Th2 cytokine (IL-4, IL-5, IL-13 and tnf- alpha) in asthmatic mice, increase the secretion of anti inflammatory factor IL-10, and reduce BALF white cells and eosinophilia. The number of acidic granulocytes alleviates infiltration of inflammatory cells and mucus secretion in lung tissue; in addition, the trimer can effectively reduce the exudation of Evans blue, reduce vascular permeability, and reduce the passive skin anaphylaxis in mice. And the antiallergic effect of the trimer is significantly stronger than that of the simple tripterin and the tripterin polymer micelles group. An anti human Fc epsilon RI alpha Fab- polymer micelle, tripterin trimer, has been successfully constructed. The trimer has a good characterization and mast cell targeting, which can inhibit mast cell proliferation and induce apoptosis in a special way. It can promote the accumulation of drugs in the lungs of allergic asthmatic mice in vivo, allergic asthma and passive skin allergic reaction in mice. These results suggest that the anti human Fc epsilon RI alpha Fab- polymer micelle - tripterin trimer has potential application value in the treatment of allergic and mast cell related diseases.

【學位授予單位】：上海交通大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R593.1

【參考文獻】

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1 李超;伏圣博;劉華玲;馬欣榮;;細胞凋亡研究進展[J];世界科技研究與發(fā)展;2007年03期

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本文編號：1808051