抗人FcεRIα Fab-聚合物胶束-雷公藤红素诱导肥大细胞凋亡治疗过敏性疾病

发布时间：2018-04-26 23:02

本文选题：雷公藤红素 + 聚合物胶束　；参考：《上海交通大学》2015年博士论文

【摘要】：研究目的:构建偶联抗人FcεRIαFab包裹雷公藤红素的聚合物胶束(抗人FcεRIαFab-聚合物胶束-雷公藤红素三聚物),分析其对肥大细胞的靶向性、增殖抑制及促凋亡作用,探讨其在过敏性哮喘小鼠体内的分布及对哮喘和被动皮肤过敏反应的抑制效果。研究方法:(1)采用薄膜水化法制备雷公藤红素-聚合物胶束,EDC偶联法构建抗人FcεRIαFab-聚合物胶束-雷公藤红素三聚物,采用粒度分析仪、透射电子显微镜分别观察胶束的粒径和形态,反相-高效液相色谱法测定其包封率和载药量。(2)体外以人外周血嗜碱性白血病细胞KU812(表面高表达FcεRI,靶细胞)和人未成熟肥大细胞HMC-1(表面几乎不表达FcεRI,非靶细胞)为研究对象,以香豆素6为探针,采用流式细胞仪和激光共聚焦显微镜检测三聚物的靶向作用;CCK-8法测定细胞增殖抑制率,Annexin V-FITC/PI双标记合并流式细胞仪定量分析凋亡细胞比率,酶法和免疫印迹分别检测凋亡分子caspase 3活性和切割的PARP表达水平,分析三聚物对肥大细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,以近红外荧光素dir为探针,观察三聚物在过敏性哮喘小鼠体内的分布及其对肺组织的靶向性;给予三聚物干预治疗,elisa法检测血清ova特异性ige(ova-sige)和肺泡灌洗液(balf)组胺的水平,采用luminex技术测定balf中th1/th2细胞因子的含量,计数balf白细胞总数及嗜酸性粒细胞数,观察肺组织炎症情况,分析三聚物对过敏性哮喘的治疗效果。此外,通过皮内注射ige-dnp和尾静脉注射dnp-hsa诱导小鼠被动皮肤过敏反应,给予三聚物干预治疗,观察伊文氏蓝扩散程度,验证三聚物对过敏反应的抑制作用。研究结果:(1)制备了抗人fcεriαfab-聚合物胶束-雷公藤红素三聚物,包封率为98.9±3.3%,载药量为22.8±1.0%,粒径在93.43nm左右,分布均匀,呈短棒状或椭圆状。(2)流式细胞仪和激光共聚焦显微镜检测结果显示抗人fcεriαfab-聚合物胶束进入靶细胞的量显著高于未偶联抗体的胶束;细胞增殖和凋亡的结果表明抗人fcεriαfab-聚合物胶束-雷公藤红素三聚物作用组的肥大细胞抑制率、凋亡细胞比例、caspase3活性和切割的parp水平均显著高于单纯的雷公藤红素和雷公藤红素-聚合物胶束组。(3)体内研究结果表明,三聚物能促进药物在过敏性哮喘小鼠的肺组织聚集,并有效地减少哮喘小鼠血清ova-sige、balf组胺和th2细胞因子(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白细胞和嗜酸性粒细胞数量,减轻肺组织炎细胞浸润和黏液分泌;此外三聚物能有效减少伊文氏蓝的渗出,降低血管通透性,减轻小鼠被动皮肤过敏反应。且三聚物的抗过敏效果明显比单纯的雷公藤红素和雷公藤红素-聚合物胶束组更强。研究结论:成功构建了抗人FcεRIαFab-聚合物胶束-雷公藤红素三聚物,该三聚物具有良好的表征和肥大细胞靶向性,能特异地抑制肥大细胞增殖、诱导其凋亡;在体内能促进药物在过敏性哮喘小鼠肺部聚集,对小鼠过敏性哮喘和被动皮肤过敏反应具有明显的抑制作用。这些结果提示抗人FcεRIαFab-聚合物胶束-雷公藤红素三聚物具有治疗过敏及肥大细胞相关疾病的潜在应用价值。
[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.

【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R593.1

【参考文献】