學習筆記-懸空七少-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-??B), 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 C?? (PLC??), 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 PLC??/PKC pathways mediated calcium influx, and promotes the generation of inflammatory mediators via NF-??B pathway resulting in anaphylactoid reaction

RBL-2H3細胞不適合用于類過敏模型的建立

目的考察RBL-2H3細胞是否適用于建立類過敏反應模型。方法用熒光定量聚合酶鏈反應考察RBL-2H3細胞上Mas相關G蛋白偶聯(lián)受體（Mas-related G protein coupled receptor, Mrgpr）B2的表達情況;用顯微鏡觀察和MTS法考察Compound 48/80對RBL-2H3細胞活力的影響;測定不同濃度Compound 48/80刺激RBL-2H3細胞脫顆粒釋放的β-己糖胺酶含量,對比RBL-2H3細胞、人肥大細胞系LAD2和大鼠腹腔肥大細胞（rat peritoneal mast cells, RPMCs）對Compound 48/80響應性的差異。結果 RBL-2H3細胞可表達MrgprB2受體。Compound 48/80能劑量依賴性地誘導RBL-2H3細胞釋放β-己糖胺酶,但在高劑量(≥20 mg·L-1)時對RBL-2H3的細胞活力產(chǎn)生明顯影響,此時釋放的β-己糖胺酶應當是由于其細胞毒作用引起細胞破裂所致。同在無毒劑量(10 mg·L-1)的Compound 48/80刺激下, LAD2和RPMCs的響應性良好,β-己糖胺酶釋放量分別為空白對照的15.02倍和16.05倍,而RBL-2H3細胞僅為空白對照的2.35倍。結論 RBL-2H3細胞對Compound 48/80的響應性差,表明其并不適用于建立類過敏反應模型。

近十余年，速發(fā)型超敏反應性疾病的患病率迅速增高，已成為當今社會的流行病，這與環(huán)境因素和生活方式的改變有關。速發(fā)型超敏反應主要分為免疫介導型和非免疫介導型兩大類。免疫介導型中最為常見的是由IgE-FcεRI介導的I型速發(fā)型超敏反應，也就是常說的過敏反應。其發(fā)生是由于過敏原初次進入機體誘導B細胞產(chǎn)生IgE抗體，抗體以Fc段與靶細胞表面FcεRI結合，使機體處于致敏狀態(tài)。當同一過敏原再次進入機體，與致敏靶細胞表面IgE抗體結合，使FcεRI交聯(lián)活化，誘導靶細胞脫顆粒，釋放活性介質(zhì)。而非免疫型超敏反應，國內(nèi)學者常稱之為類過敏反應，其誘發(fā)機制主要有兩類：一是通過直接方式刺激效應細胞(主要是肥大細胞)，例如基礎分泌素(P物質(zhì)、Compound 48/80和黃蜂毒素等)可直接結合肥大細胞表面的Mas相關的G蛋白偶聯(lián)受體(Mas-related G protein coupled receptor，Mrgpr)，致使細胞活化引起脫顆粒，同時釋放生物活性介質(zhì)[1]；二是通過間接的方式刺激肥大細胞釋放生物活性介質(zhì)，如雙黃連注射劑可通過活化補體毒素C5最終引起速發(fā)型超敏反應[2]。

大鼠腹腔肥大細胞的提取

異氟烷麻醉健康大鼠，股動脈放血致死。腹腔注射20 mL預冷的Hank’s平衡緩沖鹽溶液，輕柔按摩腹部3 min后，剪開腹部皮膚，沿腹白線剖開腹部，將腹腔內(nèi)容物推向一側，以滴管將腹腔液收集于離心管中，放入冰浴內(nèi)冷卻，600×g離心10 min，棄上清得肥大細胞沉淀物，用Hank’s平衡緩沖鹽溶液重懸得肥大細胞混懸液。

吐溫 80 誘導 RBL-2H3 細胞脫顆粒作用研究

通過觀察吐溫 80 對 RBL-2H3 細胞的誘導脫顆粒作用, 以初步探討吐溫 80 導致類過敏反應的可能機制。 采用不同 濃度的吐溫 80 溶液與 RBL-2H3 細胞共孵育, ELISA 和生化法測定過敏活性物質(zhì)組胺和β 氨基己糖苷酶釋放率 , Annexin V-FITC 熒光抗體染色法(流式細胞儀檢測和熒光顯微鏡觀察)檢測細胞膜磷脂酰絲氨酸外翻情況;結果 250 、 1 250、 6 250 mg/ L 吐溫 80 溶液均可以導致 RBL-2H3 細胞組胺和β 氨基己糖苷酶釋放率增加(P <0 .05 或 P <0.01), 呈濃度依賴性;流 式細胞儀檢測和熒光顯微鏡觀察發(fā)現(xiàn)此 3 個濃度吐溫 80 可以導致 RBL-2H3 細胞膜磷脂酰絲氨酸外翻增加(P <0 .01), 且 呈濃度依賴性;使用含鈣與不含鈣緩沖體系進行孵育。 結果顯示, β 氨基己糖苷酶釋放率沒有顯著性差異(P >0 .05)。 吐溫 80 可以在不依賴細胞外游離鈣離子的情況下直接誘導肥大細胞脫顆粒, 釋放活性介質(zhì), 這可能是其導致類過敏反應的重要 原因之一。

? ? 組胺是肥大細胞、 嗜堿粒細胞活化脫顆粒后釋 放的組氨酸經(jīng)組氨酸脫羧酶脫羧后產(chǎn)生的活性物 質(zhì), 是經(jīng)典的肥大細胞脫顆粒標志性物質(zhì) 。β 氨基 己糖苷酶(Hex)也是一種儲存在肥大細胞分泌顆粒 中的活性物質(zhì) 。研究表明, 當肥大細胞活化脫顆粒 時β 氨基己糖苷酶的釋放與組胺平行, 因此目前國 外研究中也常用此指標做為肥大細胞活化脫顆粒的 標志[ 8] 。研究結果顯示, 吐溫 80 與 RBL-2H3 細 胞共孵育后, 細胞的組胺釋放率和β 氨基己糖苷酶 釋放率均明顯上升 , 且呈濃度依賴性關系 , 說明吐 溫 80 可以不經(jīng)過免疫系統(tǒng)的復雜調(diào)節(jié)網(wǎng)絡而直接 激活肥大細胞 , 誘導其脫顆粒, 釋放活性介質(zhì) , 從 而產(chǎn)生一系列過敏反應癥狀 。研究中 β 氨基己糖苷 酶與組胺釋放率相一致 , 而生化法測定β 氨基己糖 苷酶較組胺測定更為簡便經(jīng)濟, 因此可能更適合做 為肥大細胞脫顆粒的標志性物質(zhì) 。

目前 , 研究認為肥大細胞脫顆粒依賴于胞質(zhì)中 游離鈣離子濃度的增加[ 13] , 在 IgE 介導的肥大細 胞脫顆粒過程中細胞外的鈣離子內(nèi)流起著十分重要 的作用, 耗竭細胞外游離鈣離子, 則脫顆粒反應也 將被終止[ 14] 。而本研究結果顯示 , 吐溫 80 各濃度 的含鈣與不含鈣組比較, β 氨基己糖苷酶釋放率均 沒有顯著性差異 , 說明耗竭反應體系中的游離鈣離 子對吐溫 80 誘導的 RBL-2H3 細胞脫顆粒作用沒 有明顯影響 , 吐溫 80 對肥大細胞的直接誘導脫顆 粒作用是不依賴于細胞外游離鈣離子的 , 此與經(jīng)典 IgE 抗體介導的肥大細胞脫顆粒作用機制不同 。

綜上所述, 吐溫 80 可以在不依賴細胞外游離 鈣離子的情況下直接誘導肥大細胞脫顆粒, 釋放活 性介質(zhì), 這可能是其導致類過敏反應的重要原因之 一, 其誘導機制尚有待進一步研究 。

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.