P物质,血管活性肠肽和神经肽Y在大鼠舌肥大细胞内的定位研究

采用甲苯胺蓝染色和免疫组织化学ＡＢＣ方法，对大鼠舌组织中肥大细胞的分布，形态特点以及细胞内的Ｐ物质、血管活性肠肽和神经肽Ｙ的定位进行了研究。结果表明，大鼠舌组织内肥大细胞主要分布于固有层和肌层，多呈圆形，椭圆形或长梭形。经与甲苯胺蓝染色的相邻切片比较，几乎所有肥大细胞分别呈Ｐ物质、血管活性肠肽和神经肽Ｙ免疫反应阳性。提示：舌组织肥大细胞内Ｐ物质，血管活性肠肽和神经肽Ｙ等生物活性肽可能与其局部血液供     

小鼠下颔下腺中肥大细胞异质性研究

取小鼠下颌下腺,用甲苯胺蓝染色、Alcian蓝一藏红染色及免疫组织化学ABC法显示肥大细胞的异质性.结果表明;肥大细胞主要分布于间质的小血管、小叶间导管及神经节周围.此外还发现有些肥大细胞沿腺实质表面排列,有些肥大细胞伸出突起与相邻的神经元或肥大细胞接触.肥大细胞呈降钙素基因相关肽免疫反应阳性,但仅为相邻切片甲苯胺蓝染色的14 %.提示下颌下腺中肥大细胞的存在可能与腺体分泌活动及血流调节有关.     

小鼠下颌下腺中肥大细胞异质性研究

取小鼠下颌下腺，用甲苯胺蓝染色Ａｌｃｉａｎ蓝－藏红染色及免疫组织化学ＡＢＣ法显示大细胞的异质性。结果表明：肥大细胞主要分布于间的小血管，小叶间导管及神经节周围。此外否定还发现有些肥大细胞沿腺实质表面排列，有些肥大细胞突起与相邻的神经元或肥大细胞接触。     

介绍一种显示肥大细胞快捷简便的染色方法

目前关于肥大细胞的染色方法很多,如经典的甲苯胺蓝染色、阿尔辛蓝-番红花红染色、硫堇染色、地衣红染色等,因种种原因目前尚无法广泛应用,本文现介绍一种显示肥大细胞的染色方法,该方法安全、简便、快捷,并且可用于新鲜或固定的组织标本。     

小鼠淋巴结内肥大细胞生长抑素免疫组织化学观察

目的：观察小鼠淋巴结内肥大细胞的分布特点及神经肽性质。方法：采用甲苯胺蓝染色和免疫组织化学ABC方法。结果：淋巴结内肥大细胞多为圆形和椭圆形，主要分布于淋巴窦内；经与甲苯胺蓝邻片比较，肥大细胞呈生长抑素（SS）免疫反应性，可见免疫反应阳性颗粒充满于胞质内，细胞核为阴性反应。结论：小鼠淋巴结肥大细胞呈SS免疫反应性，表明肥大细胞内神经肽SS可能通过神经－内分泌－免疫网络系统调节免疫器官的功能活动。     

肥大细胞在辐射后器官纤维化中的作用

用新建立的阿里新蓝－番红花红双染法，抗５－溴脱氧尿苷单抗和图像分析技术，研究肥大细胞在正常和照射大鼠组织内的分布特点及其颗粒活性介质（组胺）对成纤维细胞生长的影响，结果表明了肥大细胞及其亚群在组织内不均匀分布，辐射后的变化趋势以及与辐射后器官纤维化的关系，提示了肥大细胞重要颗粒活性介质－组胺，在辐射后器官纤维化的发生中可能起着不可忽视的作用。     

P物质和VIP免疫反应在人皮肤内经络线上肥大细胞颗粒内定位的研究

本研究用光镜和电镜结合的PAP技术,研究了人皮肤内足阳明经络线上P物质和血管活性肠肽(VIP)的免疫组织化学反应。发现皮肤内一部分肥大细胞的颗粒有P物质样物质,同时肯定了某些肥大细胞的颗粒有VIP样物质。观察到P物质免疫反应阳性的细胞多在网状浅层,其数量仅为甲苯胺蓝染色的肥大细胞数的3-7%。由于对照切片均阴性,排除了假阳性的可能。所用的Somagyi和Takagyi固定液比Bouin氏改良液更适于肥大细胞颗粒内这两种肽的免疫染色,前者比后者固定的标本免疫反应强,定位清楚。     

肥大细胞在平滑肌细胞源性泡沫细胞形成中的作用

目的 研究肥大细胞(或肥大细胞裂解上清液)、平滑肌细胞与氧化低密度脂蛋白(ox LDL)共孵育时,肥大细胞对平滑肌细胞源性泡沫细胞形成的影响.方法 用ox LDL处理大鼠腹腔肥大细胞后,采用荧光分光光度仪检测组胺释放率,通过倒置显微镜、甲苯胺蓝染色、透射电镜观察肥大细胞脱颗粒状态.将肥大细胞(或肥大细胞裂解上清液)、平滑肌细胞与60 mg/L ox LDL 共育48 h,以油红O染色观测细胞内中性脂质,高效液相色谱法检测细胞内胆固醇和胆同醇酯含量.结果 肥大细胞被ox LDL 激活脱颗粒,肥大细胞脱颗粒度与ox LDL呈剂鼍依赖性关系,以60 mg/L ox LDL处理肥大细胞时,肥大细胞脱颗粒度达71.70%±3.02%;甲苯胺蓝染色和透射电镜均显示,ox LDL处理后肥大细胞明显脱颗粒.在60 mg/L ox LDL存在时,以肥大细胞(或肥大细胞裂解上清液)处理平滑肌细胞48 h后,油红O染色显示,处理组胞浆内脂滴明显多于未处理组;高效液相色谱分析结果 显示,肥大细胞裂解上清液处理组细胞内总胆固醇和胆固醇酯含量明显高于未处理组,总胆固醇增高了 0.57倍,胆固醇酯增高了0.83倍.结论 ox LDL 以剂量依赖性方式诱导肥大细胞脱颗粒;肥大细胞脱颗粒后促进ox LDL诱导的平滑肌细胞源性泡沫细胞形成.     

红鲫肥大细胞鉴别方法比较

目的探讨红鲫(Cyprinus carpio)肥大细胞(MC)的不同鉴别方法。方法取已被迟缓爱德华菌感染的红鲫鳃组织,用Bouin氏液固定3 h,苏木精-伊红(HE)法、甲苯胺蓝(TB)法、阿利新蓝(AB)法、中性红法、Masson三色法、May-Grünwald Giemsa(MGG)法与免疫组织化学SABC法观察MC。同时使用Wright-Giemsa染色的头肾涂片法作为补充。结果阿利新蓝和甲苯胺蓝法显示肥大细胞良好,MGG法次之。免疫组织化学法检测类胰蛋白酶阳性肥大细胞数量较少,阳性反应弱。涂片法可将细胞分散开,并显示出不同发育阶段肥大细胞的特性。结论组织化学染色结合涂片法和免疫组织化学染色,可以更好的识别鱼类肥大细胞。     

大鼠膝关节软骨不同染色方法的差异

背景:国内外学者对关节软骨对软骨进行了大量研究,需要用不同的染色方法对研究结果进行分析,但将多种染色方法同时应用于软骨研究及探讨染色机制的较少。目的:探讨不同染色方法对大鼠膝关节软骨染色的优缺点。方法:取正常大鼠膝关节软骨,行苏木精-伊红、番红O、阿尔辛蓝、甲苯胺蓝、番红-阿尔辛蓝、番红-固绿染色,观察软骨结构。结果与结论:苏木精-伊红、番红O、甲苯胺蓝染色均可观察到潮线,分别为蓝色、红色和蓝色,番红O和甲苯胺蓝染色强度朝潮线方向增强,番红O染色对潮线的观察优于其他染色方法。苏木精-伊红染色关节软骨4层结构清晰,软骨细胞呈柱状排列,基质呈均匀呈嗜碱性染色。番红O染色显示4层结构层次清楚,基质深层染色最红。阿尔辛蓝染色显示,pH1.0时软骨细胞周边部分被阿尔辛蓝强烈染色,pH2.5时阿尔辛蓝染色较深。甲苯胺蓝染色显示组织结构层欠清,胞核染色清晰,胞浆几乎不着色,基质呈淡蓝紫色。番红-阿尔辛蓝染色显示软骨表面及基质呈不均一的红色,深层颜色较深,软骨细胞周围蓝染;番红-固绿染色显示软骨基质呈均匀的红色,软骨下骨呈绿色,软骨组织与骨组织分对比鲜明。提示上述各种方法均可观察到软骨的四层结构,但以番红O染色显示软骨各层和潮线结构最佳;苏木精-伊红染色观察软骨细胞形态变化较其他方法清楚。     

Vasoactive intestinal peptide stimulates immunoglobulin production and growth of human B cells.

The effect of vasoactive intestinal peptide (VIP) on human lymphoblastoid B cell lines and tonsil B cells was studied. VIP increased immunoglobulin production and proliferation by lymphoblastoid B cell line, GM-1056, in a dose-dependent manner. As little as 10(-12) M of VIP was effective, and higher concentrations of VIP induced an approximately five-fold increase in IgA production. Moreover, this enhancement was blocked by VIP antagonist. Similarly, VIP enhanced IgM and IgG production by other lymphoblastoid B cell lines, CBL and IM-9, respectively. In contrast to VIP, another neuropeptide substance P (SP) or somatostatin failed to enhance immunoglobulin production and thymidine uptake. VIP also enhanced IgA production and thymidine uptake by purified tonsil B cells. However, in contrast to B cell lines, VIP failed to enhance IgM and IgG production by tonsil B cells. SP or somatostatin failed to enhance immunoglobulin production or thymidine uptake by tonsil B cells. These results indicate that VIP acts as B cell stimulatory factor and that VIP may also have preferential effect on IgA production on tonsil B cells.     

中华眼镜蛇毒金属蛋白酶诱导人肥大细胞释放组胺的作用

目的： 探索中华眼镜蛇毒金属蛋白酶(MT)对人肥大细胞释放组胺的诱导作用及其机制。 方法： 用肝素凝胶和Superdex75亲和力凝胶纯化MT。将手术切除的人肺、大肠和扁桃体组织在37 ℃用Ⅰ型胶原酶和Ⅰ型透明质酸酶消化, 悬浮细胞均匀地加入含MT、刺激剂或缓冲液的试管中,进行激发实验并用荧光显色法测量上清液的组胺水平。 结果： 纯化后的MT在SDS-PAGE上呈1条带。MT能诱导人肺、大肠和扁桃体肥大细胞发生剂量依赖性组胺释放。浓度低至0.03 mg/L时, MT就能诱导大肠肥大细胞显著性的组胺释放, 但对于肺和扁桃体的肥大细胞，MT的最低有效浓度分别为0.3 mg/L和30 mg/L。 MT诱导大肠和肺肥大细胞组胺释放，12 min时达高峰，而扁桃体肥大细胞的组胺释放则在8 min时达高峰。人大肠、肺和扁桃体肥大细胞经代谢抑制剂和百日咳毒素预处理后，MT诱导其释放组胺的作用明显减弱。缺乏外源性钙、镁离子时，MT诱导肥大细胞释放组胺的能力也明显减弱。 结论： MT可能通过G蛋白偶联受体途径激活人肥大细胞，从而参与毒蛇咬伤人体后所发生的病理生理过程。     

In vitro effects of H1-antihistamines on histamine and PGD2 release from mast cells of human lung, tonsil, and skin

Mast cells from different anatomic sites differ in cytochemistry and response to various secretory stimuli. We have investigated whether responsiveness to the second-generation H₁-receptor antagonists, which are important first-line drugs for the relief of symptoms in patients with chronic urticaria and allergic rhinoconjunctivitis, also differs according to the site of origin of mast cells. The effects of terfenadine, ketotifen, and cetirizine were therefore examined in relation to the IgE-dependent release of histamine and prostaglandin D₂ (PGD₂) from dispersed human lung, tonsil, and skin mast cells. Terfenadine had a biphasic effect on lung and skin mast cells: at low concentrations, a concentration-dependent inhibition of histamine release from lung and skin mast cells was observed, whereas at higher concentrations the drug stimulated mediator release. Even at a high concentration, terfenadine inhibited mediator release from tonsil mast cells. Ketotifen had low potency as an inhibitor of mediator release from lung and tonsil mast cells. In skin mast cells, no inhibition of mediator release was observed below 1.0 μM, and above that concentration it induced mediator release. Cetirizine, a much less lipophilic drug than the others tested, did not induce mediator release from mast cells even at concentrations up to 100 μM. This drug showed concentration-dependent inhibition of IgE-dependent mediator release from lung and tonsil mast cells only. Our results show that human mast cells are heterogeneous with respect to modulation of mediator release by these H₁-antihistamines. In particular, differences were observed between skin mast cells and those dispersed from lung and tonsils.     

Neuropeptides activate human mast cell degranulation and chemokine production

During neuronal-induced inflammation, mast cells may respond to stimuli such as neuropeptides in an FcepsilonRI-independent manner. In this study, we characterized human mast cell responses to substance P (SP), nerve growth factor (NGF), calcitonin gene-related peptide (CGRP) and vasoactive intestinal polypeptide (VIP) and compared these responses to human mast cell responses to immunoglobulin E (IgE)/anti-IgE and compound 48/80. Primary cultured mast cells, generated from CD34(+) progenitors in the presence of stem cell factor and interleukin-6 (IL-6), and human cultured mast cells (LAD2) were stimulated with these and other stimuli (gastrin, concanavalin A, radiocontrast media, and mannitol) and their degranulation and chemokine production was assessed. VIP and SP stimulated primary human mast cells and LAD cells to degranulate; gastrin, concanavalin A, radiocontrast media, mannitol, CGRP and NGF did not activate degranulation. While anti-IgE stimulation did not induce significant production of chemokines, stimulation with VIP, SP or compound 48/80 potently induced production of monocyte chemoattractant protein-1, inducible protein-10, monokine induced by interferon-gamma (MIG), RANTES (regulated on activation, normal, T-cell expressed, and secreted) and IL-8. VIP, SP and compound 48/80 also activated release of tumour necrosis factor, IL-3 and granulocyte-macrophage colony-stimulating factor, but not IL-4, interferon-gamma or eotaxin. Human mast cells expressed surface neurokinin 1 receptor (NK1R), NK2R, NK3R and VIP receptor type 2 (VPAC2) but not VPAC1 and activation of human mast cells by IgE/anti-IgE up-regulated expression of VPAC2, NK2R, and NK3R. These studies demonstrate the pattern of receptor expression and activation of mast cell by a host of G-protein coupled receptor ligands and suggest that SP and VIP activate a unique signalling pathway in human mast cells. These results are likely to have direct relevance to neuronally induced inflammatory diseases.     

Down-regulation of vasoactive intestinal polypeptide receptor expression in atopic dermatitis

BACKGROUND: Receptors for vasoactive intestinal polypeptide (VIP) have recently been suggested to play a key role in immunomodulation with genetically modified mice. However, it is not known whether changes in receptor gene regulation are involved in the pathogenesis of human immune disorders. OBJECTIVE: We studied the expression of VPAC(2) in acute lesions of the human immune disease atopic dermatitis. METHODS: By using nonradioactive in situ hybridization, quantitative immunohistochemistry, RT-PCR, and gene array studies, the expression status of VPAC(2) was assessed in atopic dermatitis and control tissues and in the human mast cell line HMC-1. RESULTS: In situ hybridization and immunohistochemistry demonstrated VPAC(2) mRNA and protein expression in human mast cells surrounded by VIP positive nerve fibers. Gene array experiments and RT-PCR studies showed high levels of VPAC(2) mRNA expression in mast cells that were increased compared to other receptors such as VPAC(1) or VIP in the human mast cell line HMC-1. Stimulation of HMC-1 cells led to a downregulation of VPAC(2). Similarly, quantitative immunohistochemistry for VPAC(2) in acute atopic dermatitis lesions showed a significantly decreased VPAC(2) immunoreactivity in mast cells. CONCLUSION: The downregulation of VPAC(2) in human mast cells in acute lesions of atopic dermatitis suggests a role of this G-protein;coupled receptor in the pathophysiology of the disease.     

特异性类胰蛋白酶抑制剂双苯甲脒对肥大细胞分泌的调控作用

目的：研究新型的特异性类胰蛋白酶抑制剂双苯甲脒，对肥大细胞稳定性的影响。方法：扁桃体组织经酶消化后，将细胞成分用全HEPES缓冲盐溶液（HBSS）重新悬浮。肥大细胞激发和抑制剂作用的试验在试管中37℃条件下完成，类胰蛋白酶水平用ELISA法测定。结果：同时加入扁桃体细胞悬液中的双苯甲脒，可以剂量依赖的方式抑制抗IgE诱导的类胰蛋白酶释放，仅1mg/L(1.54μmol/L）的双杠甲脒，即可抑制肥大细胞释放类胰蛋白酶达52%，10mg/L则能抑制76%的释放，将预培养时间延长30min，对双苯甲脒的抑制作用无明显影响，在培养到45min时，双苯甲脒可抑制高达47%的基础类胰蛋白酶释放，与细胞培养15min后，抗IgE抗体（1g/L)或钙离子导入剂（CI，1μmol/L）引起的类胰蛋白酶释放的量，分别为基础分泌量的3.2和2.6倍，但是，当细胞与全HBSS预培养超过10min后，肥大细胞对抗IgE抗体或CI的反应性明显降低。结论：双苯甲脒能够抑制人类扁桃体肥大细胞的IgE依赖性类胰蛋白酶释放。因素，有望开发成为一种新型的肥大细胞稳定剂。     

大鼠舌组织中肥大细胞与肽能神经纤维的分布

目的探讨舌组织内肥大细胞与肽能神经的关系及其功能意义。方法采用甲苯胺蓝染色和免疫组织化学ABC染色法对10只Wistar大鼠舌组织进行SP、VIP和NPY染色。结果经与甲苯胺蓝邻片对比观察,舌组织内肥大细胞分别呈SP、VIP和NPY免疫反应性;在舌组织固有层及肌间结缔组织中还分布有丰富的SP、VIP和NPY免疫反应阳性纤维;在肽能神经纤维附近或周围可见有免疫反应阳性的肥大细胞,这些肥大细胞与神经纤维紧密相靠或接触。结论 大鼠舌组织内肥大细胞和神经纤维分别呈SP、VIP和NPY免疫反应性,提示肥大细胞与周围神经无论是在形态还是在功能上有着密切的联系。     

QUANTITATIVE HISTOCHEMICAL ANALYSIS OF MAST CELLS AND SENSORY NERVES IN PSORIATIC SKIN

To study the elements of neurogenic inflammation in psoriatic skin, morphological contacts were examined between mast cells and sensory nerves containing the neuropeptides substance P (SP), calcitonin gene-related peptide (CGRP) or vasoactive intestinal polypeptide (VIP). Because mast cells in psoriatic lesions appear in great numbers at the basement membrane (BM) zone, neuropeptide–mast cell contacts with the BM were also counted. A double stain for active mast cell tryptase and the neuropeptides was applied and the contacts were quantitated morphometrically. Sensory nerve–mast cell contacts were also studied three-dimensionally with a confocal laser scanning microscope. Increases in the contact values of SP and CGRP with mast cells, as well as with the BM, were obtained in developing (1–3 weeks) lesions when compared with their non-lesional controls. This increase reached statistical significance in mature lesions. In contrast, the corresponding contact values for VIP were decreased. By confocal microscopy, a close association between mast cells and sensory nerves was observed in the lesional dermis. Since tryptase is known to degrade CGRP but not SP, neurogenic stimuli, mainly via SP, can result in degranulation of mast cells, which release substances to enhance inflammation. At the BM zone in psoriatic lesions, the numerous mast cells loaded with tryptase can promote degradation of BM components and allow entry of various mediators to interact with keratinocytes.     

Immunolocalization of tyrosine hydroxylase and vasoactive intestinal polypeptide in nerve fibers innervating human palatine tonsil and paratonsillar glands

The presence of tyrosine hydroxylase (TH) and vasoactive intestinal polypeptide (VIP) in the nerve fibers of the human palatine tonsil and paratonsillar secretory glands is reported. By immunohistochemistry TH-immunoreactive nerves and those immunoreactive to VIP were localized to the tonsil, in particular, the tonsillar vessel wall, extranodular lymphoid tissue and lymph nodule, and to the acinar basal surface of the paratonsillar glands. In the lymph nodule, immunoreactive varicose nerve profiles were observed inside the marginal zone. The germinal center was devoid of immunoreactive fibers.