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

【摘要】：研究目的:構(gòu)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(duì)肥大細(xì)胞的靶向性、增殖抑制及促凋亡作用,探討其在過(guò)敏性哮喘小鼠體內(nèi)的分布及對(duì)哮喘和被動(dòng)皮膚過(guò)敏反應(yīng)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(gòu)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(cè)定其包封率和載藥量。(2)體外以人外周血嗜堿性白血病細(xì)胞KU812(表面高表達(dá)FcεRI,靶細(xì)胞)和人未成熟肥大細(xì)胞HMC-1(表面幾乎不表達(dá)FcεRI,非靶細(xì)胞)為研究對(duì)象,以香豆素6為探針,采用流式細(xì)胞儀和激光共聚焦顯微鏡檢測(cè)三聚物的靶向作用;CCK-8法測(cè)定細(xì)胞增殖抑制率,Annexin V-FITC/PI雙標(biāo)記合并流式細(xì)胞儀定量分析凋亡細(xì)胞比率,酶法和免疫印跡分別檢測(cè)凋亡分子caspase 3活性和切割的PARP表達(dá)水平,分析三聚物對(duì)肥大細(xì)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(guò)敏性哮喘小鼠體內(nèi)的分布及其對(duì)肺組織的靶向性;給予三聚物干預(yù)治療,elisa法檢測(cè)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(shù)測(cè)定balf中th1/th2細(xì)胞因子的含量,計(jì)數(shù)balf白細(xì)胞總數(shù)及嗜酸性粒細(xì)胞數(shù),觀察肺組織炎癥情況,分析三聚物對(duì)過(guò)敏性哮喘的治療效果。此外,通過(guò)皮內(nèi)注射ige-dnp和尾靜脈注射dnp-hsa誘導(dǎo)小鼠被動(dòng)皮膚過(guò)敏反應(yīng),給予三聚物干預(yù)治療,觀察伊文氏藍(lán)擴(kuò)散程度,驗(yàn)證三聚物對(duì)過(guò)敏反應(yīng)的抑制作用。研究結(jié)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(xì)胞儀和激光共聚焦顯微鏡檢測(cè)結(jié)果顯示抗人fcεriαfab-聚合物膠束進(jìn)入靶細(xì)胞的量顯著高于未偶聯(lián)抗體的膠束;細(xì)胞增殖和凋亡的結(jié)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(xì)胞抑制率、凋亡細(xì)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(nèi)研究結(jié)果表明,三聚物能促進(jìn)藥物在過(guò)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(xì)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(xì)胞和嗜酸性粒細(xì)胞數(shù)量,減輕肺組織炎細(xì)胞浸潤(rùn)和黏液分泌;此外三聚物能有效減少伊文氏藍(lán)的滲出,降低血管通透性,減輕小鼠被動(dòng)皮膚過(guò)敏反應(yīng)。且三聚物的抗過(guò)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(qiáng)。研究結(jié)論:成功構(gòu)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(xì)胞靶向性,能特異地抑制肥大細(xì)胞增殖、誘導(dǎo)其凋亡;在體內(nèi)能促進(jìn)藥物在過(guò)敏性哮喘小鼠肺部聚集,對(duì)小鼠過(guò)敏性哮喘和被動(dòng)皮膚過(guò)敏反應(yīng)具有明顯的抑制作用。這些結(jié)果提示抗人FcεRIαFab-聚合物膠束-雷公藤紅素三聚物具有治療過(guò)敏及肥大細(xì)胞相關(guān)疾病的潛在應(yīng)用價(jià)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(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R593.1

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

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

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