特异性脱颗粒肥大细胞的形态学研究

目的 观察特异性脱颗粒肥大细胞的形态特征。方法 通过抗ＩｇＥ单抗（抗ＩｇＥ ｍＡｂ，ＨＲＰ）与已致敏的肥大细胞相结合，用ＤＡＢ加中胜红双染色法对肥大细胞进行染色。结果 发现无论肥大细胞是否脱颗粒，只有被ＩｇＥ致敏的肥大细胞才能被染成棕褐色；而非特异性（即未被ＩｇＥ致敏的肥大细胞），无论其脱颗粒与否，都呈现中性红的颜色，即胞浆内呈现砖红色的颗粒。结论 该实验为评价患者当前所处状态（是高敏体质还是非高敏体质？是疾病缓解期抑或发作期等？）、预测哮喘等Ⅰ型变态反应的病程转归、判断疗效，以及进行一些与肥大细胞脱颗粒有关的科学实验，提供了一种新的更客观的方法。     

肥大细胞脱颗粒促进外源树突状细胞向引流淋巴结归巢

目的:探讨通过诱导局部肥大细胞脱颗粒促进外源骨髓源性树突状细胞(BM-DC)向淋巴结迁移归巢的方法。方法:采用肥大细胞脱颗粒刺激剂compound 48/80(c48/80)进行C57BL/6小鼠局部皮肤注射,诱导局部组织肥大细胞脱颗粒。将体外培养的荧光微球标记的同系小鼠BM-DC注射入c48/80或生理盐水处理的局部皮肤,48h收集注射部位引流淋巴结细胞进行CD11c荧光抗体染色,流式细胞术测定外源回输的BM-DC的迁移效果。结果:c48/80注射小鼠皮肤后可有效地促进局部组织肥大细胞脱颗粒,c48/80处理侧局部引流淋巴结中外源BM-DC迁移细胞总数和淋巴结细胞总数较对照侧均明显增多,增多比例分别为67%±43%和55%±43%(P0.05)。结论:通过诱导局部皮肤肥大细胞脱颗粒,可促进外源BM-DC向淋巴结归巢。     

百日咳鲍特菌外膜蛋白体外促肥大细胞脱颗粒的作用

目的了解百日咳鲍特菌Ⅰ相菌株外膜蛋白(outer membrane proteins,OMP)生物学活性与IgE 介导哮喘的关系.方法采用去垢剂处理、DEAE-52和Sephadex G-150层析法制备百日咳鲍特菌Ⅰ相菌株OMP及其组分,以SDS-PAGE估计OMP组分的相对分子质量(Mr).采用ELISA检测百日咳全菌死疫苗诱导动物产生的总IgE和卵白蛋白(ovalbumin,OVA)特异的IgE量.采用肥大细胞脱颗粒试验观察OMP及其组分促进脱颗粒的作用、ELISA检测培养物上清中组胺水平.采用组胺敏感试验检测OMP及其组分增加小鼠对组胺致死敏感性的作用.结果从百日咳鲍特菌OMP中分离获得Mr为(30、32、38和69)×103的4个组分.1～100μg/ml OMP及其组分均未显示凝集鲎变形细胞溶解物的活性.OVA和百日咳鲍特菌联合免疫豚鼠的血清总IgE和OVA特异性IgE较单用OVA免疫有明显增加(t=2.75～6.59;P<0.05～0.01).OMP及其Mr 30×103、Mr 32×103组分有明显的促豚鼠肥大细胞脱颗粒的作用(t=4.914～9.137,P<0.01),但Mr 38×103、Mr 69×103组分无此活性.OMP及其Mr 30×103、Mr 32×103组分处理的上清中组胺水平明显高于阴性对照组(t=5.672～9.304,P<0.01),但Mr 38×103、Mr 69×103组分无此活性.结论百日咳鲍特菌有较强的IgE佐剂活性,某些OMP组分具有促进肥大细胞脱颗粒、组胺释放及增加小鼠对组胺致死敏感性的作用,提示百日咳鲍特菌感染或疫苗接种与IgE介导的哮喘有密切关系.     

木犀草素对肥大细胞脱颗粒影响及机制的研究

目的:探讨木犀草素(Luteolin)的抗I 型变态反应的作用机制。方法:通过建立DNP-BSA-IgE 激发致敏的大鼠RBL-2H3 细胞模型,分别采用MTT 法检测不同浓度(5、15、25 mol/ L)Luteolin 对RBL-2H3 肥大细胞活性的影响;ELISA 法检测不同浓度Luteolin 对RBL-2H3 细胞分泌 hexosa minidase( HEX)及细胞因子TNF 影响;Flou-4AM 荧光探针检测细胞内Ca2+浓度变化;Western blot 检测AKT,P-AKT 的表达。结果:成功建立致敏的细胞模型,低浓度的Luteolin 对RBL-2H3 细胞活性无明显影响;不同浓度Luteolin 刺激RBL-2H3 细胞后,对 HEX 和TNF鄄琢的释放抑制作用呈线性相关,且细胞内Ca2+ 明显减少;Western blot 结果显示随着Luteolin 浓度的增加AKT 磷酸化水平明显下降。结论:Luteolin 呈剂量依赖性抑制RBL鄄2H3 肥大细胞脱颗粒,且通过调节细胞内Ca2+浓度与AKT 活性参与其中。     

肥大细胞脱颗粒信号的自我放大机制

目的：变态反应性疾病包括支气管哮喘、变应性鼻炎、变应性皮炎、食物及药物过敏等，发病率约占全球人口的40％，对人类危害极大。目前已经证实此类疾病的发生是一种炎症过程，而肥大细胞被认为在这种炎症过程中起着重要的作用，被称为初级效应细胞（primary effector cells），是连接变态反应诱导阶段和效应阶段的纽带。但是，迄今为止微量过敏原引起机体大量肥大细胞激活的机制尚不清楚，因而很有必要给以深入研究。方法：①肥大细胞激活实验：以类胰蛋白酶、组胺、蛋白酶激活受体激动剂等激活酶悬浮的人大肠肥大细胞，并分别用酶联免疫吸附试验和特异性玻璃纤维结合荧光检测技术测量类胰蛋白酶和组胺的分泌量②小白鼠腹腔炎症性细胞聚集实验：注射后收集腹腔灌洗液并分类计数炎症性细胞③应用激光共聚焦显微镜结合双色荧光标记鉴定肥大细胞亚型。     

TNBS诱导结肠炎小鼠中细菌鞭毛蛋白的表达及其与肥大细胞脱颗粒的关系

目的:研究不同炎症程度下2,4,6-三硝基苯磺酸(TNBS)诱导的结肠炎小鼠的血清及结肠组织中细菌鞭毛蛋白CBir1的表达、肥大细胞脱颗粒的情况及两者的相关性。方法:将SPF级雄性BALB/c小鼠随机分为6组,每组12只,分别是:正常对照组、生理盐水组、50%乙醇组、50%乙醇+TNBS组、50%乙醇+TNBS+酮替芬组及50%乙醇+TNBS+脂多糖(LPS)+卵清蛋白(OVA)组,同时对小鼠进行疾病活动指数(DAI)评分。分组处理后第22天处死并提取小鼠的血清及结肠组织,采用组织损伤指数(HI)评分标准对小鼠结肠进行组织病理评估,采用ELISA法检测抗-CBir1、组胺以及肥大细胞类胰蛋白酶(MCT)在小鼠血清中的浓度,并用免疫组化法检测小鼠结肠组织中CBir1、Toll样蛋白受体5(TLR5)及MCT的表达。结果:TNBS诱导组的DAI评分和HI评分,且CBir1、TLR5、MCT、抗-CBir1和组胺在肠黏膜和血清中的表达明显高于正常对照组(P0.05);50%乙醇+TNBS组上述数值除TLR5外均低于50%乙醇+TNBS+LPS+OVA组(P0.05);50%乙醇+TNBS组上述数值除MCT外则均高于50%乙醇+TNBS+酮替芬组(P0.05);生理盐水和50%乙醇组小鼠与正常对照组小鼠比较,上述数值差异无统计学显著性。将TNBS诱导模型组小鼠血清中抗-CBir1与MCT的浓度进行相关性分析,结果提示两者呈正相关(r=0.751,P0.01);此外,小鼠的血清中抗-CBir1与组胺的浓度亦呈正相关(r=0.648,P0.01)。结论:TNBS诱导结肠炎炎症程度越重的小鼠,其体内CBir1的表达量越高,同时肥大细胞脱颗粒作用也越明显。TNBS诱导结肠炎小鼠体内CBir1的表达量与肥大细胞脱颗粒作用呈正相关性。     

激光诱发荧光在结肠肿瘤早期诊断中的应用

激光诱导荧光法 (L IF)利用生物组织的自体荧光特性来判断组织性质 ,能实时、无损地提供组织信息 ,从而区分正常与病变组织。本文详述了生物组织自体荧光谱技术应用于结肠肿瘤诊断的研究方法、最新成果和现状 ,并对其发展和应用提出了几点建议。     

改良的人嗜碱细胞脱颗粒试验在花粉变态反应研究中的应用

改良的人嗜碱细胞脱颗粒试验是一种观察人嗜碱细胞对特异性变应原脱颗粒反应的简易方法。其步骤包括聚蔗糖——泛影葡胺浓缩嗜碱细胞,用阿尔新蓝特异性染色细胞浆内肝素颗粒,借助于血球计数板计数未脱粒细胞,以加入特异性变应原后染色细胞减少30％以上判定为阳性。本文以蒿属花粉作为特异性变应原,对50例由蒿属花粉过敏所致的典型秋季花粉症患者作嗜碱细胞脱颗粒试验。结果发现,在花粉症缓解期阳性率为72％,且其阳性率随蒿属花粉皮试反应强度的增加而升高,而在花粉症急性发作期阳性率为34％,明显低于缓解期。由此对“嗜碱细胞释放能力”这一重要参数进行了初步探讨,认为在花粉症急性发作期嗜碱细胞在体内已经或正在和特异性变应原发生作用,此时如果嗜碱细胞在体外再和变应原接触时,由于其释放能力的限制,将处于相对的低反应状态。30例正常人的阴性反应结果则排除了蒿属花粉变应原的非特异性脱颗粒作用。本研究表明该方法在花粉变态反应研究中应用的敏感性和稳定性。     

Human mast cell tryptase in biology and medicine

The most abundant prestored enzyme of human mast cell secretory granules is the serine-protease tryptase. In humans, there are four tryptase isoforms, but only two of them, namely the alpha and beta tryptases, are known as medically important. Low levels of continuous tryptase production as an immature monomer makes up the major part of the baseline serum tryptase levels, while transient release of mature tetrameric tryptase upon mast cell degranulation accounts for the anaphylactic rise of serum tryptase levels. Serum tryptase determination contributes to the diagnosis or monitoring of mast cell disorders including mast cell activation – induced anaphylaxis, mastocytosis and a number of myeloproliferative conditions with mast cell lineage involvement. Baseline serum tryptase levels are predictive of the severity risk in some allergic conditions.     

Induction of mast cell degranulation by triterpenoidal saponins obtained from Cimicifugae rhizoma

Cimicifugae rhizoma has been widely used as a traditional herbal medicine to treat inflammation and menopausal symptoms. In this study, we found that some of the triterpenoidal saponins purified from the ethanol extract of Cimicifugae rhizoma dramatically induced histamine release. The structure-related induction of mast cell degranulation by them and the mechanism of action were determined. β-Hexosaminidase release in HMC-1 cells was increased in a concentration-dependent manner, with maximal 6.5- and 8.5-fold increases, by 200?μg/mL 24-epi-7,8-didehydrocimigenol-3-O-xyloside (comp 1) and cimigenol 3-O-beta-d-xyloside (comp 4) compared with those treated with phorbol 12-myristate 13-acetate and A23187 (PMACI), respectively. However, β-hexosaminidase release was not changed by 7,8-dihydrocimigenol (comp 3), or 23-OAc-shengmanol-3-O-xyloside (comp 7). These triterpenoidal saponins changed neither the intracellular Ca^2+?level nor the activation of PKC, both of which play essential roles in histamine release. However, cromolyn and ketotifen, membrane stabilizers, effectively inhibited the β-hexosaminidase release induced by comp 1 or comp 4 by 39 and 45%, respectively. Collectively, xylose on the cimigenol-related backbone among triterpene glycosides isolated from Cimicifugae rhizoma may play an important role in activating mast cells and induction of degranulation partly via membrane destabilization of mast cells.     

Single-cell dynamics of mast cell-CD4+ CD25+ regulatory T cell interactions

The biological behavior of immune cells is determined by their intrinsic properties and interactions with other cell populations within their microenvironment. Several studies have confirmed the existence of tight spatial interactions between mast cells (MCs) and Tregs in different settings. For instance, we have recently identified the functional cross-talk between MCs and Tregs, through the OX40L-OX40 axis, as a new mechanism of reciprocal influence. However, there is scant information regarding the single-cell dynamics of this process. In this study, time-lapse video microscopy revealed direct interactions between Tregs and MCs in both murine and human cell co-cultures, resulting in the inhibition of the MC degranulation response. MCs incubated with WT, but not OX40-deficient, Tregs mediated numerous and long-lasting interactions and displayed different morphological features lacking the classical signs of exocytosis. MC degranulation and Ca2+ mobilization upon activation were inhibited by Tregs on a single-cell basis, without affecting overall cytokine secretion. Transmission electron microscopy showed ultrastructural evidence of vesicle-mediated secretion reconcilable with the morphological pattern of piecemeal degranulation. Our results suggest that MC morphological and functional changes following MC-Treg interactions can be ascribed to cell-cell contact and represent a transversal, non-species-specific mechanism of immune response regulation. Further research, looking at the molecular composition of this interaction will broaden our understanding of its contribution to immunity.     

The effect of ulinastation on the small intestine injury and mast cell degranulation in a rat model of sepsis induced by CLP

IntroductionSepsis could be initiated by the gastrointestinal tract injury and subsequent bacterial translocation. In the present experiment, we aimed to investigate effect of ulinastatin (UTI) on the small intestinal injury and bacterial translocation in septic rats and role of mast cells degranulation in its action.MethodsFifty-four male Wistar rats were randomly divided into three groups: sham laparatomy, cecal ligation and punture (CLP), and CLP plus UTI. CLP was used to develop septic rat model and UTI was administered to rats intraperitoneally (50,000 U/kg) 30 min prior to CLP operation. After CLP or sham operation, variable parameters were investigated in three subsets of animals. One subset was used for measurements of nitrite and nitrate (NO_x) concentration in plasma at 1, 6, 12, 18, and 24 h and levels of NO_x and iNOS mRNA in the small intestine, RMCP-II released into the small intestinal lumen, bacterial translocation and morphologic changes at 24 h. The other subsets were used for the small intestinal motility and microvascular in vivo at 24 h.ResultsBacterial translocation, barrier injury, impaired motility and blood flow, mast cells degranulation of the small intestine in the CLP group were found more severe than that in the sham group. Elevated RMCP-II, NO_x, and iNOS mRNA levels were also detected in the CLP group. Application of UTI not only protected the small intestine from sepsis but also diminished changes of intestinal mast cells.ConclusionUTI can significantly ameliorate the small intestinal injury and subsequent bacterial translocation by inhibiting mast cells degranulation in septic rats.     

Recombinant human alpha-2a interferon promotes an atypical process of mast cell secretion with ultrastructural features suggestive for piecemeal degranulation

Purified mast cells (MC), isolated from the rat peritoneum, were stimulated in vitro with recombinant human IFN-alpha 2a (rhIFN-alpha 2a) and studied ultrastructurally. Quantitative determination of histamine release was also performed. The following ultrastructural features were observed: (1) dilation of single granules, leading to the formation of cytoplasmic cavities filled with dissolved or eroded matrices; (2) induction of partially empty, nonfused granule containers close to unaltered resting granules, a process very suggestive of piecemeal degranulation; (3) focal exocytosis, characterized by opening of single granules to the cell exterior and/or fusion of a few granules into small secretory channels. Histamine release was slightly increased in rhIFN-alpha 2a-treated MC, although not to significant levels. These results indicate that rhIFN-alpha 2a induces a characteristic pattern of degranulation in rat peritoneal MC and that a small proportion of rhIFN-alpha 2a-stimulated MC shows ultrastructural features suggestive for piecemeal degranulation.     

Osteopontin is produced by mast cells and affects IgE-mediated degranulation and migration of mast cells

Osteopontin (OPN), originally discovered in bone as an extracellular matrix protein, was identified in many cell types in the immune system, presumably being involved in many aspects of pathogenesis of inflammatory and immune diseases. Mast cells are also involved in such pathological aspects by secreting multiple mediators. However, it has not been determined whether mast cells produce OPN and whether it affects their function. To test this, we used murine fetal skin-derived cultured mast cells (FSMC) and bone marrow-derived cultured mast cells. We found that OPN was spontaneously produced by FSMC and inducible by ionomycin and FcepsilonRI aggregation in bone marrow-derived cultured mast cells. In the presence of mast cell growth factors, FSMC were similarly generated from both OPN-deficient (OPN(-/-)) and -sufficient (OPN(+/+)) mice without significant differences in yield, purity, granularity, and viability. Using OPN(-/-) FSMC, we found that recombinant OPN augmented IgE-mediated degranulation and induced FSMC chemotaxis. Both effects were mediated by OPN receptors (i.e. CD44 and integrin alphav). IgE-mediated passive cutaneous anaphylaxis was significantly reduced in OPN(-/-) mice compared with OPN(+/+) mice, indicating physiological relevance of OPN. These results indicate that OPN is a mast cell mediator, enhances mast cell responses to antigen, and thus may influence mast cell-related pathological conditions.     

Histamine modulates mast cell degranulation through an indirect mechanism in a model IgE-mediated reaction

Histamine is released in inflammatory reactions and exerts an immunoregulatory function on cells present in the microenvironment. In this study, we compared the effect of histamine on degranulation of mast cells derived from animals bearing a parasitic infection with those from uninfected animals. Peritoneal mast cells (PMC) were obtained 24 days after infection of Wistar rats with Toxocara canis. The degree of degranulation was assessed either morphologically or by measuring the release of beta-hexosaminidase and TNF-alpha. Non-purified PMC or mast cells immunomagnetically purified with mAb AA4 were used. An increase in degranulation of non-purified mast cells from infected animals was observed after incubation with histamine in vitro or when histamine was injected into the peritoneal cavity. When a purified mast cell population was used, this effect was no longer observed. Supernatants from spleen cells stimulated with histamine induced degranulation of purified mast cells, and again, this was potentiated with PMC from infected animals. However, when supernatants from peritoneal macrophages similarly stimulated were used, a reduction in the degranulation of PMC from infected animals was observed. Our results suggest that histamine may act as a regulator of mast cell degranulation, thus modulating inflammatory responses due to infection with certain parasites.     

天花粉蛋白对肥大细胞脱颗粒和组胺释放的影响及其与补体激活的关系

我们首先用Alcian蓝染色的肥大细胞脱颗粒试验和用荧光测定组胺的方法观察了天花粉蛋白对肥大细胞脱颗粒和组胺释放的影响。实验结果表明:(1)在体外试验中,不论有无血清的存在,天花粉蛋白都不引起肥大细胞的脱粒和组胺释放。(2)天花粉蛋白的体内用药,能引起Balb/c小鼠炎症局部的肥大细胞脱颗粒(Alcian着色的未脱颗粒肥大细胞由对照组的3.4±0.12×10~4/ml减少至1.7±0.28×10~4/l;P<0.01)和组胺水平的明显增高(皮下气囊内的组胺水平由36.39±0.94ng/ml升至41.07±0.78ng/ml;P<0.01)。(3)腹膜腔内注射天花粉蛋白后,Wistatr大鼠腹膜腔内蛋白明显渗出,同时全血组胺水平也明显增高(对照组:0.947±0.076ng/ml,实验组:1.574±0.105ng/ml;p<0.01)。(4)上述天花粉蛋白诱导的腹腔渗出液具有明显的肥大细胞脱颗粒作用(脱粒指数:对照组为1.6±3.8%,实验组为24.9±2.1%;P<0.01)。(5)用眼镜蛇毒素完全耗竭小鼠的补体对天花粉蛋白引起的肥大细胞脱粒因子的产生无明显影响。从这些结果可见:天花粉蛋白的体内应用能通过激活某些可溶性成分的产生而引起肥大细胞的脱颗粒和组胺释放。但是,这一过程与天花粉蛋白激活补体的作用似无明显关系。