【菜鸟博士学习】20200511类过敏细胞模型学习
学习笔记-悬空七少-Caesar
Label-Free Quantitative Proteomic Profiling of LAD2 Mast Cell Releasates Reveals the Mechanism of Tween-80-Induced Anaphylactoid Reaction
Purpose: Tween-80 is one of the most important causes resulting in anaphylactoid reaction. However, its mechanism remains unclear. Proteomic characterizations of mast cells’ excreta in response to Tween-80 are assayed to investigate the mechanism of anaphylactoid reaction. Experimental design: A label-free LCMS/MS-based proteomics is used to analyze Tween-80-stimulated Laboratory of Allergic Diseases 2 (LAD2) mast cells releasates. The results of proteomic are analyzed by bioinformatics analysis. Western blotting is used to verify the expression of proteins. Results: Overall, endocytosis, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-𝜿B), and calcium signaling pathways play important roles in Tween-80-induced LAD2 cells activation by bioinformatics analysis. The expressions of relative proteins including actin-related protein 2/3 complexes, vacuolar protein sorting-associated protein, phosphorylation of transcription factor of P105 and P65, phosphorylation of inositol 1,4,5-trisphosphate receptor (IP3R), phosphoinositide phospholipase C𝜸 (PLC𝜸), and protein kinase C (PKC), are significantly increased in Tween-80 group compared to control. Tween-80 might be internalized via endocytosis, which induces degranulation by PLC𝜸/PKC pathways mediated calcium influx, and promotes the generation of inflammatory mediators via NF-𝜿B pathway resulting in anaphylactoid reaction
RBL-2H3细胞不适合用于类过敏模型的建立
目的考察RBL-2H3细胞是否适用于建立类过敏反应模型。方法用荧光定量聚合酶链反应考察RBL-2H3细胞上Mas相关G蛋白偶联受体（Mas-related G protein coupled receptor, Mrgpr）B2的表达情况;用显微镜观察和MTS法考察Compound 48/80对RBL-2H3细胞活力的影响;测定不同浓度Compound 48/80刺激RBL-2H3细胞脱颗粒释放的β-己糖胺酶含量,对比RBL-2H3细胞、人肥大细胞系LAD2和大鼠腹腔肥大细胞（rat peritoneal mast cells, RPMCs）对Compound 48/80响应性的差异。结果 RBL-2H3细胞可表达MrgprB2受体。Compound 48/80能剂量依赖性地诱导RBL-2H3细胞释放β-己糖胺酶,但在高剂量(≥20 mg·L-1)时对RBL-2H3的细胞活力产生明显影响,此时释放的β-己糖胺酶应当是由于其细胞毒作用引起细胞破裂所致。同在无毒剂量(10 mg·L-1)的Compound 48/80刺激下, LAD2和RPMCs的响应性良好,β-己糖胺酶释放量分别为空白对照的15.02倍和16.05倍,而RBL-2H3细胞仅为空白对照的2.35倍。结论 RBL-2H3细胞对Compound 48/80的响应性差,表明其并不适用于建立类过敏反应模型。
近十余年，速发型超敏反应性疾病的患病率迅速增高，已成为当今社会的流行病，这与环境因素和生活方式的改变有关。速发型超敏反应主要分为免疫介导型和非免疫介导型两大类。免疫介导型中最为常见的是由IgE-FcεRI介导的I型速发型超敏反应，也就是常说的过敏反应。其发生是由于过敏原初次进入机体诱导B细胞产生IgE抗体，抗体以Fc段与靶细胞表面FcεRI结合，使机体处于致敏状态。当同一过敏原再次进入机体，与致敏靶细胞表面IgE抗体结合，使FcεRI交联活化，诱导靶细胞脱颗粒，释放活性介质。而非免疫型超敏反应，国内学者常称之为类过敏反应，其诱发机制主要有两类：一是通过直接方式刺激效应细胞(主要是肥大细胞)，例如基础分泌素(P物质、Compound 48/80和黄蜂毒素等)可直接结合肥大细胞表面的Mas相关的G蛋白偶联受体(Mas-related G protein coupled receptor，Mrgpr)，致使细胞活化引起脱颗粒，同时释放生物活性介质[1]；二是通过间接的方式刺激肥大细胞释放生物活性介质，如双黄连注射剂可通过活化补体毒素C5最终引起速发型超敏反应[2]。
大鼠腹腔肥大细胞的提取
异氟烷麻醉健康大鼠，股动脉放血致死。腹腔注射20 mL预冷的Hank’s平衡缓冲盐溶液，轻柔按摩腹部3 min后，剪开腹部皮肤，沿腹白线剖开腹部，将腹腔内容物推向一侧，以滴管将腹腔液收集于离心管中，放入冰浴内冷却，600×g离心10 min，弃上清得肥大细胞沉淀物，用Hank’s平衡缓冲盐溶液重悬得肥大细胞混悬液。
吐温 80 诱导 RBL-2H3 细胞脱颗粒作用研究
通过观察吐温 80 对 RBL-2H3 细胞的诱导脱颗粒作用, 以初步探讨吐温 80 导致类过敏反应的可能机制。 采用不同 浓度的吐温 80 溶液与 RBL-2H3 细胞共孵育, ELISA 和生化法测定过敏活性物质组胺和β 氨基己糖苷酶释放率 , Annexin V-FITC 荧光抗体染色法(流式细胞仪检测和荧光显微镜观察)检测细胞膜磷脂酰丝氨酸外翻情况;结果 250 、 1 250、 6 250 mg/ L 吐温 80 溶液均可以导致 RBL-2H3 细胞组胺和β 氨基己糖苷酶释放率增加(P <0 .05 或 P <0.01), 呈浓度依赖性;流 式细胞仪检测和荧光显微镜观察发现此 3 个浓度吐温 80 可以导致 RBL-2H3 细胞膜磷脂酰丝氨酸外翻增加(P <0 .01), 且 呈浓度依赖性;使用含钙与不含钙缓冲体系进行孵育。 结果显示, β 氨基己糖苷酶释放率没有显著性差异(P >0 .05)。 吐温 80 可以在不依赖细胞外游离钙离子的情况下直接诱导肥大细胞脱颗粒, 释放活性介质, 这可能是其导致类过敏反应的重要 原因之一。
组胺是肥大细胞、 嗜碱粒细胞活化脱颗粒后释 放的组氨酸经组氨酸脱羧酶脱羧后产生的活性物 质, 是经典的肥大细胞脱颗粒标志性物质 。β 氨基 己糖苷酶(Hex)也是一种储存在肥大细胞分泌颗粒 中的活性物质 。研究表明, 当肥大细胞活化脱颗粒 时β 氨基己糖苷酶的释放与组胺平行, 因此目前国 外研究中也常用此指标做为肥大细胞活化脱颗粒的 标志[ 8] 。研究结果显示, 吐温 80 与 RBL-2H3 细 胞共孵育后, 细胞的组胺释放率和β 氨基己糖苷酶 释放率均明显上升 , 且呈浓度依赖性关系 , 说明吐 温 80 可以不经过免疫系统的复杂调节网络而直接 激活肥大细胞 , 诱导其脱颗粒, 释放活性介质 , 从 而产生一系列过敏反应症状 。研究中 β 氨基己糖苷 酶与组胺释放率相一致 , 而生化法测定β 氨基己糖 苷酶较组胺测定更为简便经济, 因此可能更适合做 为肥大细胞脱颗粒的标志性物质 。
目前 , 研究认为肥大细胞脱颗粒依赖于胞质中 游离钙离子浓度的增加[ 13] , 在 IgE 介导的肥大细 胞脱颗粒过程中细胞外的钙离子内流起着十分重要 的作用, 耗竭细胞外游离钙离子, 则脱颗粒反应也 将被终止[ 14] 。而本研究结果显示 , 吐温 80 各浓度 的含钙与不含钙组比较, β 氨基己糖苷酶释放率均 没有显著性差异 , 说明耗竭反应体系中的游离钙离 子对吐温 80 诱导的 RBL-2H3 细胞脱颗粒作用没 有明显影响 , 吐温 80 对肥大细胞的直接诱导脱颗 粒作用是不依赖于细胞外游离钙离子的 , 此与经典 IgE 抗体介导的肥大细胞脱颗粒作用机制不同 。
综上所述, 吐温 80 可以在不依赖细胞外游离 钙离子的情况下直接诱导肥大细胞脱颗粒, 释放活 性介质, 这可能是其导致类过敏反应的重要原因之 一, 其诱导机制尚有待进一步研究 。
Evaluation of anaphylactoid constituents in vitro and in vivo
Natural medicine injections have been widely used in clinics, while adverse reaction reports also have increased rapidly in recent years. To examine the anaphylactoid constituents of natural medicine injections, RBL-2H3 cell degranulation and human serum complement activation models were used to screen the anaphylactoid constituents, and the BN rat model was used to explore the anaphylactoid mechanism of these constituents. The result of an in vitro study showed that the individual compounds of natural medicine injections (chlorogenic acid, hyodeoxycholic acid, cholalic acid, ginkgolic acid, phillyrin, schisandrin B, schisandrin A, puerarin, and tanshinone IIA) and polysaccharide could not induce RBL-2H3 to release histamine and β-hexosaminidase, while proteins Tween-80 and tannic acid were the main anaphylactoid constituents in the natural medicine injections. The in vivo study also indicated that > 10 kDa molecules (proteins) activated classical complement pathways through direct stimulation to cause an anaphylactoid reaction. Tween-80 activated direct stimulation and coagulation pathways through classical and alternative pathways; tannic acid induced anaphylactoid reaction through co-activation of the kallikrein-kinin system, coagulation, integrated, classical and alternative complement pathways. This is the first study to evaluate the anaphylactoid constituents systematically through in vitro and in vivo study. And tannic acid, > 10 kDa molecules (proteins), and injection additives such as Tween-80 are the main anaphylactoid constituents of natural medicine injections.
