参麦注射液致RBL-2H3细胞脱颗粒的研究

目的 探讨参麦注射液(SMI)对RBL-2H3细胞脱颗粒的影响及其原因。方法 以C48/80为工具药建立RBL-2H3细胞脱颗粒模型, 检测不同浓度C48/80与RBL-2H3细胞作用不同时间后细胞β-己糖苷酶、类胰蛋白酶和组胺的释放率以及细胞活力, 在细胞活力大于80%情况下, 选择释放程度较高的指标和条件为优选考察指标和条件。将SMI和其溶剂(Tween-80)原液等比稀释成不同浓度后与RBL-2H3细胞共同培养, 通过中性红染色法观察细胞脱颗粒的形态学变化, 分别用显色法和间接荧光法检测细胞上清的β-己糖苷酶和组胺释放率, 采用细胞计数法(CCK-8)检测细胞的活力。结果 RBL-2H3细胞脱颗粒模型的最佳作用时间为30 min, 最佳指标为β-己糖苷酶和组胺释放率。与空白组相比, SMI质量浓度低于13.3 g生药/L(3倍临床浓度)时, 细胞中性红染色未见脱颗粒现象, 组胺和β-己糖苷酶释放率亦无差异;而在Tween-80质量浓度为1.00 g/L时, SMI 40 g生药/L(9倍临床浓度)组和溶剂1.00 g/L组细胞中性红染色均可见脱颗粒现象, 细胞上清组胺和β-己糖苷酶释放率亦明显增加。此外, CCK-8结果显示, 与空白组相比, 各浓度的SMI对细胞活力均无影响。结论 SMI低于3倍临床浓度无明显RBL-2H3细胞脱颗粒作用;而在9倍临床浓度能刺激细胞脱颗粒, 这种脱颗粒作用可能与所含溶剂(Tween-80)有关, 与其对RBL-2H3的细胞毒性作用无关。提示SMI在低于3倍临床浓度相对安全, 在9倍临床浓度时有致类过敏反应的风险。     

药用注射辅料聚山梨酯80诱发类过敏反应的细胞研究

目的:观察聚山梨酯80诱导大鼠嗜碱性粒细胞白血病细胞株RBL-2 H3细胞脱颗粒现象,探讨其诱发类过敏反应的可能机制。方法:聚山梨酯80与RBL-2 H3细胞共同孵育60 min后,透射电镜观察细胞发生脱颗粒反应的形态学变化,荧光显微镜下观察细胞膜磷脂酰丝氨酸与Annexin V结合变化,分光光度法测定细胞上清液β-氨基己糖苷酶释放百分率,并通过阿利新蓝染色试验进行定性观察。结果:不同浓度聚山梨酯80可诱导细胞发生典型的脱颗粒反应形态学变化,与阴性对照组相比脱颗粒率及组胺释放率显著升高,且具有浓度依赖性。结论:聚山梨酯80单次、首次给药即可诱导RBL-2 H3细胞脱颗粒,释放组胺,诱发类过敏反应,这可能是其临床首次用药即引起严重不良反应的机制之一。     

中药注射剂所含吐温-80与过敏反应关系的研究

目的：观察不同浓度吐温-80溶液、吐温-80含量不同的中药注射剂对RBL-2H3细胞脱颗粒的影响,探讨中药注射剂所含吐温-80与过敏反应的关系。方法：体外培养RBL-2H3细胞,加入不同浓度（40、20、10、2、1、0.2、0.1、0.05mg/mL）的吐温-80溶液,之后加中性红染液,计数不同浓度吐温-80溶液各组及对照组的脱颗粒细胞,并计算其百分率,同时检测细胞上清液中β-氨基己糖苷酶及组胺的释放量;测定穿琥宁注射液和香丹注射液中吐温-80的含量,2种中药注射剂对RBL-2H3细胞的半数抑制浓度（IC50）以及加入2种注射液各组细胞释放组胺的量。结果：中性红染色实验显示吐温-80可导致RBL-2H3细胞脱颗粒,表现为肥大细胞体积变大,内有空泡产生;浓度为40、20、10、2、1、0.2、0.1mg/mL的吐温-80溶液各组和RPMI1640对照组导致细胞的脱颗粒百分率分别为（57.38±0.47）、（32.54±2.33）、（21.74±0.72）、（16.96±0.26）、（11.40±1.70）、（9.71±0.26）、（7.22±0.15）和（1.51±1.39）%,2组相比差异有统计学意义（P〈0.05,P〈0.01）;浓度为40、20、2、1、0.2mg/mL的吐温-80溶液各组和RPMI1640对照组致细胞β-氨基己糖苷酶的释放率分别为（52.44±1.53）、（18.91±0.77）、（7.50±1.82）、（6.65±0.20）、（6.15±0.27）和（0.35±0.06）%,2组相比差异有统计学意义（P〈0.05,P〈0.01）;不同浓度吐温-80溶液引起RBL-2H3细胞释放组胺的量也不同;当吐温-80溶液浓度为20～0.1mg/mL时,RBL-2H3细胞脱颗粒百分率、β-氨基己糖苷酶的释放率及其释放组胺的量均与吐温-80溶液的浓度呈线性关系（r=0.9862,r=0.9849,r=0.9740）。穿琥宁注射液和香丹注射液中吐温-80的含量分别为（0.086±0.004）和（0.070±0.008）mg/mL,2种注射液对RBL-2H3细胞的IC50分别为（57.4±1.2）、（1.0±0.2）μL/mL,穿琥宁注射液和香丹注射液组组胺释放量分别为（2.39±0.01）和（1.87±0.00）ng/mL。结论：吐温-80可引起RBL-2H3细胞脱颗粒释放炎症介质;RBL-2H3细胞组胺的释放量与中药注射剂中吐温-80的含量有关;中药注射剂中所含的吐温-80可能与过敏反应的发生有关。     

非免疫性过敏反应细胞模型的建立

目的:通过非免疫性刺激物C48/80诱导大鼠嗜碱性粒细胞白血病细胞株RBL-2H3细胞脱颗粒反应正交试验,优化非免疫性过敏反应细胞模型建立条件.方法:在不同的孵育时间下,C48/80与不同浓度的RBL-2H3细胞共孵育,通过测定β-氨基己糖苷酶的释放率确定建立非免疫型过敏反应细胞模型的最优实验条件,并且分析不同检测方法间的差异.结果:不同浓度、不同孵育时间下的RBL-2H3细胞均可受C48/80诱导发生典型的脱颗粒反应,但不同条件下的脱颗粒程度和β-氨基己糖苷酶释放率都有显著差异.结论:当RBL-2H3细胞浓度为2×105 mL、孵育时间60 min时,细胞的感受性较好,其脱颗粒程度与药物浓度呈正相关.     

虾原肌球蛋白及其IgE单克隆抗体建立I型过敏反应体内外模型

目的利用虾原肌球蛋白及其Ig E单克隆抗体建立I型过敏反应体内外模型。方法采用等电点沉淀法制备刀额新对虾原肌球蛋白,利用杂交瘤技术制备抗虾原肌球蛋白Ig E单克隆抗体。建立虾原肌球蛋白攻击Ig E单抗致敏的RBL-2H3细胞模型,测定β-氨基己糖苷酶释放量。利用虾原肌球蛋白及其Ig E单抗建立被动全身过敏反应小鼠模型,监测抗原攻击后30 min内肛温的变化。结果 Ig E单抗致敏的RBL-2H3细胞在虾原肌球蛋白攻击后发生脱颗粒,β-氨基己糖苷酶释放量明显增加。虾原肌球蛋白及其Ig E单抗诱导了小鼠被动全身过敏反应,抗原攻击后小鼠肛温平均下降约1.44℃。结论虾原肌球蛋白及其Ig E单克隆抗体成功建立了I型过敏反应体内外模型。     

清开灵注射液诱发类过敏反应作用及其机制研究

目的 研究清开灵注射液诱发类过敏反应的作用及其机制。方法 采用BALB/c小鼠和BN大鼠,观察给予清开灵注射液后小鼠皮肤血管渗透性以及大鼠平均动脉血压等类过敏指标的变化;利用RBL-2H3大鼠嗜碱性粒细胞白血病细胞,观察清开灵注射液作用后磷脂酰肌醇3-激酶（PI3K）活性以及Rac1、PAK1、LIMK1和人肌动蛋白素（Cofilin）蛋白含量的变化。结果 5 mL·kg^-1清开灵注射液即可引起小鼠以血管通透性增高为主要表现的较为轻微的类过敏反应,而7.5 mL·kg^-1和15 mL·kg^-1清开灵注射液均可引起较为严重的类过敏反应,其反应呈剂量依赖性;另外,5,7.5和15 mL·kg^-1清开灵注射液可在一定时间范围内剂量依赖性地引起大鼠平均动脉血压降低。清开灵注射液体外可诱导RBL-2H3细胞发生类过敏反应,PI3K激酶活性增加,Rac1、PAK1、LIMK1和Cofilin蛋白含量增高,且具有剂量依赖性。结论 小鼠和大鼠体内类过敏反应检查法和RBL-2H3细胞中PI3K激酶及Rac1、PAK1、LIMK1、Cofilin蛋白含量检查法可作为类过敏反应检查方法。初步认定清开灵注射液诱导类过敏反应的发生可能是基于PI3K-Rac1细胞运动信号转导通路。     

聚山梨酯80对多源肥大细胞脱颗粒的影响研究

目的 通过比较不同浓度聚山梨酯80溶液对RBL-2H3、P815、Ku812细胞脱颗粒的影响,探究聚山梨酯80的量与过敏反应的关系,进而筛选出一套灵敏、稳定的体外肥大细胞脱颗粒模型。方法 体外培养RBL-2H3、P815和Ku812细胞,测定3株细胞的生长曲线,在细胞生长对数期,以不同浓度（0.04、0.20、1.00、5.00、10.00、20.00、40.00、80.00 mg/mL）聚山梨酯80分别刺激3株细胞,采用中性红染色法显微镜下观察不同浓度聚山梨酯80溶液对3株细胞脱颗粒的影响,并计算脱颗粒百分率,同时以化学发光法分别检测组胺和β-氨基己糖苷酶释放率,以酶联免疫吸附法测定类胰蛋白酶的释放量;并进一步检测聚山梨酯80对人的肥大细胞IgE释放的影响。结果 3株肥大细胞脱颗粒模型中,各细胞的脱颗粒指标均随聚山梨酯80浓度的升高而升高。相同浓度聚山梨酯80对人源、大鼠、小鼠肥大细胞的类胰蛋白酶释放量无较大统计学差异;与RBL-2H3细胞系比较,P815细胞和组胺释放率显著降低（P<0.05、0.01）,Ku812细胞组胺释放率和β-氨基己糖苷酶释放率显著升高（P<0.05、0.01）,Ku812细胞脱颗粒最灵敏。但在实验过程中发现Ku812细胞模型稳定性、可重复性较差,而RBL-2H3细胞稳定性、可重复性均优于Ku812和P815细胞。0.04~80.00 mg/mL的聚山梨酯80溶液作用于Ku812细胞产生的IgE均低于检测限。结论 聚山梨酯80诱导的过敏反应可能是不经过IgE介导的类过敏反应,随着聚山梨酯80浓度的增大,3个细胞模型脱颗粒现象显著,相比于Ku812和P815细胞,RBL-2H3细胞更适合作为体外肥大细胞脱颗粒检测模型。     

基于实时细胞分析技术评价注射用益气复脉(冻干)类过敏反应

目的 建立中药注射剂体外类过敏反应评价方法,快速评价不同批次注射用益气复脉（冻干）类过敏反应表现。方法 体外培养嗜碱性白血病细胞株RBL-2H3细胞,选择Compound 48/80为阳性药,采用实时细胞分析（real-time cell analysis,RTCA）系统检测药物干预后引起的细胞指数（CI）值变化,并利用甲苯胺蓝、鬼笔环肽染色观察细胞形态、骨架变化,以及检测组胺和β-已糖苷酶释放量验证RBL-2H3细胞的脱颗粒情况。选择20个批次的注射用益气复脉（冻干,100 μg/mL）作用于RBL-2H3细胞,进行基于RTCA技术的类过敏反应评价。结果 阳性药Compound 48/80（20 μg/mL）能够使RBL-2H3细胞的CI值在加药后30 min内呈先快速上升后下降趋势;形态学研究发现,Compound 48/80使细胞形态和细胞骨架均发生明显改变,发生明显的脱颗粒现象;组胺和β-己糖苷酶释放实验进一步证实Compound 48/80导致炎症介质的释放,引起了明显的脱颗粒现象;提示RTCA系统可以用于快速敏感的评价RBL-2H3细胞脱颗粒。不同批次的注射用益气复脉（冻干）对RBL-2H3细胞CI值无明显影响,提示所选批次为合格批次,无类过敏反应现象的发生。结论 建立了一套基于RTCA系统的类过敏反应体外快速评价技术,可用于注射用益气复脉（冻干）等中药注射剂类过敏反应的体外快速评价。     

几种临床引发Ⅰ型过敏反应的中药注射剂致敏性再评价

目的 通过主动全身过敏试验（ASA）及被动皮肤过敏试验（PCA）对几种临床易于发生Ⅰ型过敏反应的中药注射剂的致敏性进行再评价.方法 取生理盐水、卵蛋白、清开灵注射液、生脉注射液、丹参注射液、喜炎平注射液对豚鼠进行主动全身过敏试验和大鼠被动皮肤过敏试验：（1）Elisa法检测主动全身过敏试验中采集的豚鼠血浆中IgE、组胺、类胰蛋白酶、β-氨基己糖苷酶的含量.（2）观察被动皮肤过敏试验中大鼠背部出现的蓝斑情况.结果 主动全身过敏试验中卵蛋白组、生脉注射液组、丹参注射液组豚鼠血浆中IgE、组胺及β-氨基己糖苷酶含量均高于生理盐水组;被动皮肤过敏试验中卵蛋白组、生脉注射液组、丹参注射液组可见明显蓝斑.结论 主动全身过敏试验结合被动皮肤过敏试验表明卵蛋白、生脉注射液、丹参注射液、清开灵注射液有致敏性,可引起Ⅰ型过敏反应的发生.     

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 cou-pled 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。     

CTB glycoprotein (75 kDa) inhibits IgE releasing,TNF-α and IL-6 expressed by bisphenol A in vivo and in vitro

Cudrania tricuspidata Bureau (CTB) has been used to treat allergies and inflammatory disease as folk medicine in Korea. The objective of this study is to determine whether a glycoprotein isolated from CTB (75 kDa) has a preventive potential of allergic inflammation caused by bisphenol A (BPA) in BALB/c mice and RBL-2H3 cells. Production of immunoglobulin (Ig) E and releasing of β-hexosaminidase and histamine at treatment of CTB glycoprotein (5–10 mg/kg, BW) were evaluated in mice serum. Activation of extracellular signal-regulated kinases (ERK) and p38 mitogen-activated protein kinase (MAPK), activator protein (AP)-1, expressions of pro-inflammatory cytokines, nitric oxide (NO) production and cyclooxygenase (COX)-2 were assessed in RBL-2H3 cells. In the results, CTB glycoprotein (10 mg/kg, BW) inhibited the production of IgE and releasing of β-hexosaminidase and histamine. Also, the CTB glycoprotein (100 μg/ml) blocked phosphorylation of ERK1/2 and p38 MAPK, and the activation of AP-1, while it inhibited the NO production, activities of COX-2, tumor necrosis factor (TNF)-α, and interleukin (IL)-6, but not IL-1β. Taken together, the results of this study indicated that the CTB glycoprotein modulates the expression of allergic inflammation-related factors via the suppression of MAPK/AP-1 activation.     

聚山梨酯80刺激肥大细胞RBL-2H3脱颗粒作用的评价

目的：研究不同类别及厂家的聚山梨酯80直接刺激肥大细胞RBL-2H3脱颗粒的作用,为建立完善的注射用辅料引发类过敏反应的筛选评价体系提供依据。方法：以不同剂量的聚山梨酯80处理RBL-2H3细胞,测定β-氨基己糖苷酶释放量,并通过MTT法进一步研究有脱颗粒作用的受试物对RBL-2H3细胞的毒性作用。结果：8种聚山梨酯80样品具有不同程度的直接刺激RBL-2H3细胞脱颗粒的作用,且呈浓度依赖性,其中上海试剂采购供应站提供的受试物作用极强,威尔制药口服级受试物作用较强,威尔制药A、威尔制药B、威尔制药注射级、威尔制药4个受试物作用相近,2个进口品种的作用略弱。在产生直接刺激RBL-2H3细胞脱颗粒作用的浓度下,上海试剂采购供应站的聚山梨酯80有明显的细胞毒性,并呈浓度依赖性。结论：聚山梨酯80能够直接刺激RBL-2H3细胞脱颗粒,其作用强度不同反映了产品的质量差异;上海试剂采购供应站提供的聚山梨酯80直接刺激RBL-2H3细胞脱颗粒的作用可能由其细胞毒性引起。     

Inhibitory mechanism of saponins derived from roots of Platycodon grandiflorum on anaphylactic reaction and IgE-mediated allergic response in mast cells

The purpose of this study was to investigate the protective effects of saponins isolated from the root of Platycodi Radix (Changkil saponins: CKS) anti-allergic effects in mice and mast cells. Oral administration of CKS inhibited the dinitrophenyl (DNP)–IgE antibody-induced systemic PCA reaction in mice. CKS reduced the β-hexosaminidase and histamine release from anti-DNP–IgE-sensitized RBL–2H3 cells. In addition, CKS inhibited the IgE antibody-induced increases in IL-4 and TNF-α production and expression in RBL-2H3 cells. In order to explore the inhibitory mechanism of CKS in PCA and mast cell degranulation, we examined the activation of intracellular signaling molecules. CKS suppressed DNP–IgE antibody-induced Syk phosphorylation. Further downstream, CKS also inhibited the phosphorylation of Akt and MAP kinases. Taken together, the in vivo/in vitro anti-allergic effects of CKS suggest possible therapeutic applications for this agent in allergic diseases through the inhibition of inflammatory cytokines and Syk-dependent signaling cascades.     

Houttuynia cordata water extract suppresses anaphylactic reaction and IgE-mediated allergic response by inhibiting multiple steps of FcεRI signaling in mast cells

Houttuynia cordata has been used as a traditional medicine in Korea and is known to have antioxidant, anti-cancer and anti-allergic activities. The precise effect of H. cordata, however, remains unknown. In this study, we investigated the effects of H. cordata water extract (HCWE) on passive cutaneous anaphylaxis (PCA) in mice and on IgE-mediated allergic response in rat mast RBL-2H3 cells. Oral administration of HCWE inhibited IgE-mediated systemic PCA in mice. HCWE also reduced antigen (DNP-BSA)-induced release of β-hexosaminidase, histamine, and reactive oxygen species in IgE-sensitized RBL-2H3 cells. In addition, HCWE inhibited antigen-induced IL-4 and TNF-α production and expression in IgE-sensitized RBL-2H3 cells. HCWE inhibited antigen-induced activation of NF-κB and degradation of IκB-α. To investigate the inhibitory mechanism of HCWE on degranulation and cytokine production, we examined the activation of intracellular FcεRI signaling molecules. HCWE suppressed antigen-induced phosphorylation of Syk, Lyn, LAT, Gab2, and PLC γ2. Further downstream, antigen-induced phosphorylation of Akt and MAP kinases (ERK1/2 and JNK1/2 but not p38 MAP kinase) were inhibited by HCWE. Taken together, the in vivo/in vitro anti-allergic effect of HCWE suggests possible therapeutic applications of this agent in inflammatory allergic diseases through inhibition of cytokines and multiple events of FcεRI-dependent signaling cascades in mast cells.     

Ephedrae herba (Mao) decreased histamine content in RBL-2H3 cells

The effect of Ephedrae herba (Mao) on histamine content was investigated. When rat basophilic leukemia (RBL-2H3) cells were incubated for 48 h with Mao, Mao decreased histamine content in a concentration-dependent manner. However, the ratio of released histamine to total histamine was scarcely affected by Mao treatment when RBL-2H3 cells were stimulated by ionomycin. On the other hand, the content of -hexosaminidase, another marker of degranulation, was only slightly decreased by Mao. The expression level of the active form (53 kDa) of l-histidine decarboxylase, the enzyme which synthesizes histamine, was partially suppressed by Mao. Furthermore, Mao significantly suppressed proliferation of RBL-2H3 cells in a concentration-dependent manner. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine revealed an increasing effect of Mao on cAMP level in RBL-2H3 cells. Since this result suggested that Mao may stimulate adenylate cyclase, we evaluated the effect of adenylate cyclase inhibitor SQ 22536 on Mao-induced decrease in histamine content. As a result, we showed that SQ 22536 did not have a reducing effect of Mao. From these results it is understood that Mao decreased histamine content by inhibiting cell proliferation and expression of histidine decarboxylase. However, these effects are not related to the increase in cAMP.