组胺对人结肠肥大细胞类胰蛋白酶释放的调节作用

探讨组胺对人结肠肥大细胞类胰蛋白酶释放的调节作用。经酶消化后获取人结肠组织肥大细胞,激发后行多种干预实验。用酶联免疫吸附试验法测定类胰蛋白酶。结果发现组胺可诱导人结肠肥大细胞释放类胰蛋白酶,浓度为100μg/L时组胺释放量最大,为基础值的3．5倍。浓度为10μg/L时对人肥大细胞的刺激强度与10mg/L的抗IgE抗体相似。组胺的作用从加样后10s开始,5min后完成。百日咳毒素和抗霉素A联合2．脱氧-D-葡萄糖可显著抑制组胺诱导人结肠肥大细胞释放类胰蛋白酶。100及1000μg/L的组胺与抗IgE抗体或离子载体钙同时加入细胞中,诱导类胰蛋白酶释放的能力低于组胺单独作用组。结论为组胺可激活人结肠肥大细胞,还以自身放大机制调节肥大细胞的脱颗粒过程。     

蛋白酶抑制剂对肥大细胞类胰蛋白酶分泌的影响

目的 探讨蛋白酶抑制剂和组胺对肥大细胞类胰蛋白酶分泌的影响。方法 扁桃体组织经酶消化后，细胞成份用全HBSS重新悬浮，肥大细胞激发和抑制剂作用的试验在37℃条件下完成。类胰蛋白酶水平用酶联免疫吸附试验（ELISA）方法测定。结果 鱼精蛋白具有刺激人类扁桃体肥大细胞释放类胰蛋白酶的作用，其分泌量可高达基础分泌量的5倍。高浓度TLCK和TPGK可抑制抗-IgE诱导的类胰蛋白酶释放，TLCK在有否预培养的情况下均能抑制钙离子导入剂（cal-cium ionophore,CI)诱导的类胰蛋白酶释放，而TPCK则只有在20min预培养后才能显示此作用。结论 TLCK和TPCK抑制IgE依赖性和非依赖性类胰蛋白酶释放提示具有胰蛋白酶和糜蛋白酶活性的酶参与肥大细胞的激活-分泌偶联过程。     

肝素对人肥大细胞组胺分泌的调节作用

目的　研究肝素对人肥大细胞组胺释放的影响。方法　经酶消化后的肺和扁桃体组织的细胞成份同肝素、抗人IgE抗体或钙离子导入剂共同培养 ,以特制的纤维玻璃为基础的方法测量组胺。结果　经 15和 4 5min培养 ,肝素对人肺和扁桃体组织肥大细胞组胺释放无明显影响。而将肝素与抗IgE抗体同时加入酶悬浮的肺肥大细胞中 (预培养时间为 0min) ,肝素可抑制 5 0 %的抗IgE抗体诱导的组胺释放 ,且抑制率与肝素浓度呈正相关。肝素对抗IgE抗体引起的扁桃体肥大细胞的组胺释放无抑制作用 ,对钙离子导入剂诱导的肺和扁桃体的肥大细胞组胺释放无明显影响。结论　本实验首次发现肝素可以抑制抗IgE抗体诱导的人肺肥大细胞组胺释放 ,因此在肺部的变态反应性炎症中可能起到一定的预防和治疗作用     

蛋白酶激活受体2激动剂对肥大细胞释放组胺的影响

目的 研究PAR-2激动剂(tc-LIGRLO-NH2与SLIGKV-NH2)和胰蛋白酶对结肠肥大细胞释放组胺的影响。方法 结肠组织经酶消化后,细胞成份用全HBSS重新悬浮。激发过程在LP4试管中、37℃条件下完成。组胺水平用以玻璃纤维为基础的荧光方法测定。结果PAR-2激动剂tc-LIGRLO-NH2和SLIGKV-NH2均可诱导人结肠肥大细胞剂量依赖性组胺释放。浓度为100μmol／mL时,tc-LIGRLO-NH2和SLIGKV-NH2可分别引起比基础分泌量多出2．5倍和2倍的组胺释放,而反PAR-2激动剂tc-OLRGIL-NH2和VKGILS-NH2在实验浓度高至300μmol／mL时仍对组胺释放无影响。胰蛋白酶在1．0～100μg/mL间可引起剂量相关性组胺释放,胰蛋白酶抑制剂可抑制之。PAR-2激动剂tc-LIGRLO-NH2的作用从加样后1min开始,3min后完成。细胞经过百日咳毒素和代谢抑制剂预先处理后,几乎失去了对tc-LIGRLO-NH2、SLIGKV-NH2和胰蛋白酶刺激的反应性。结论 PAR-2激动剂和胰蛋白酶是高效的组胺释放刺激剂,其在人结肠炎症性疾病中起一定的作用。     

亮肽素、TLCK和乳铁蛋白对人结肠肥大细胞类胰蛋白酶释放的影响

目的：探讨亮肽素、N-甲苯磺酰-L-赖氨酸氯甲基化酮（TLCK）和乳铁蛋白对人结肠肥大细胞释放类胰蛋白酶的影响。 方法： 人结肠组织经酶消化后，细胞成份用全HBSS重新悬浮。激发过程在LP4试管中、37 ℃ 条件下完成。类胰蛋白酶水平用酶联免疫吸附试验（ELISA）方法测定。 结果： 亮肽素、TLCK和乳铁蛋白无明显刺激人结肠肥大细胞释放类胰蛋白酶的作用。但可以剂量依赖性的方式抑制抗IgE抗体诱导的类胰蛋白酶释放，抑制范围在36.6%-47.6%。在37 ℃条件下同结肠细胞预培养20 min与无预培养相比，亮肽素和TLCK对抗IgE抗体诱导的类胰蛋白酶释放的抑制作用无明显改变。亮肽素、TLCK和乳铁蛋白均可以抑制CI诱导的类胰蛋白酶释放，抑制范围在27.1%-44.1%。在37 ℃条件下同结肠细胞预培养20 min与无预培养相比，亮肽素对CI诱导的类胰蛋白酶释放的抑制作用明显增强，TLCK则无此特点。 结论： 我们首次发现类胰蛋白酶抑制剂可抑制人结肠肥大细胞IgE依赖性和非依赖性类胰蛋白酶释放，提示类胰蛋白酶抑制剂可治疗炎症性肠病或其它肥大细胞相关疾病。     

类胰蛋白酶抑制剂对人大肠肥大细胞组胺释放的影响

目的 :研究类胰蛋白酶抑制剂 (TPI)对人大肠肥大细胞释放组胺的影响。方法 :大肠组织经胶原酶和透明质酸酶消化后 ,将细胞成分用全HBSS重新悬浮。肥大细胞在LP4试管中于37℃条件下与各种刺激剂反应 15min而完成激发过程。激发液中的组胺水平用以玻璃纤维为基础的荧光方法测定。结果 :4种TPI中 ,高浓度的亮抑酶肽素和鱼精蛋白可刺激人大肠肥大细胞释放组胺 ;而TLCK和乳铁蛋白则无明显的刺激作用。 4种TPI均可以剂量依赖的方式抑制抗IgE抗体诱导的大肠肥大细胞释放组胺。最大浓度的亮抑酶肽素(2 0 0mmol/L)、TLCK(10 0mmol/L)、乳铁蛋白 (30mmol/L)和鱼精蛋白 (10 0mg/L) ,可分别抑制 4 8.7%、36 .7%、4 0 .2 %和 34.1%的组胺释放。在 37℃条件下 ,将 4种TPI同大肠肥大细胞预培养 2 0min ,与未进行预培养相比较 ,它们对抗IgE抗体诱导的组胺释放无明显改变。 4种TPI还可抑制CI诱导的组胺释放 ,抑制范围在 2 5 %～ 32 %之间。与抗IgE抗体诱导的组胺释放则不同 ,与大肠肥大细胞预培养 2 0min ,与未进行预培养相比较 ,亮抑酶肽素和TLCK对CI诱导的组胺释放的抑制作用明显增强 ;而鱼精蛋白则无此作用。结论 :我们首次发现TPI可抑制人大肠肥大细胞IgE抗体依赖和非IgE抗体依赖的组胺释放 ,提示TPI可望成为炎症性肠     

组胺对肥大细胞激活的研究

组胺是肥大细胞和嗜碱性粒细胞内组氨酸脱羧基后产生的一种胺类物质 ,是肥大细胞和嗜碱性粒细胞脱颗粒的标志物 ,本研究通过组胺对人大肠肥大细胞进行体外干预 ,探讨组胺对人肥大细胞类胰蛋白酶释放的调节和可能的机制。结果发现 :1.组胺可诱导人大肠肥大细胞以非剂量依赖性的方式释放类胰蛋白酶 ,引起最大释放量的组胺浓度为 10 0ng/ml,比基础分泌量多出 3.5倍。组胺浓度高至 10 0 0ng/ml和 10 0 0 0ng/ml时 ,其诱导类胰蛋白酶的释放量介于组胺浓度为 10ng/ml与 10 0ng/ml之间。浓度为 10ng/ml的组胺对人肥大细胞的刺激强度与 10 μg/ml的…     

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。     

类糜蛋白酶抑制剂对人大肠肥大细胞类胰蛋白酶释放的影响

目的：研究类糜蛋白酶抑制剂对人大肠肥大细胞释放类胰蛋白酶的影响。方法：人大肠组织经酶消化后，细胞成份有全HBSS重新悬浮。激发过程在LP4试管中、37℃条件下完成。类胰蛋白酶水平用酶联免疫吸附试验（ELISA）方法测定。结果：促分泌剂抗IgE抗体和CI在培养15分钟和35分钟时可明显刺激人大肠肥大细胞类胰蛋白酶的释放，在相同实验体系中，类糜蛋白酶抑制剂ZIGPFM、TPCK和α1-抗胰蛋白酶无明显刺激人大肠肥大细胞释放类胰蛋白酶的作用。类糜蛋白酶抑制剂均可以剂量依赖性的方式抑制抗IgE抗体诱导的类胰蛋白酶的释放，最大浓度的ZIGPFM（1 μmol／ml）、TPCK（80 μmol／ml）和α1-抗胰蛋白酶（30 μmol／ml）可分别抑制37％、40％和36．6％的类胰蛋白酶释放。在37℃条件下同大肠细胞预培养20分钟与无预培养相比，ZIGPFM和TPCK对抗IgE抗体诱导的类胰蛋白酶释放的抑制作用略增强。Amastatin对抗IgE抗体诱导的类胰蛋白酶的释放无作用。类糜蛋白酶抑制剂均可以剂量依赖性的方式抑制CI诱导的类胰蛋白酶的释放，抑制范围在23％-35．3％。在37℃条件下同大肠细胞预培养20分钟与无预培养相比，ZIGPFM对CI诱导的类胰蛋白酶释放的抑制作用有增强，TPCK则无此特点。结论：类糜蛋白酶抑制剂可抑制人大肠肥大细胞IgE依赖性和非依赖性类胰蛋白酶的释放，提示类糜蛋白酶抑制剂可望成为炎症性肠病或其它肥大细胞相关疾病的一个新的治疗途径。     

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。     

Inhibition of tryptase release from human colon mast cells by histamine receptor antagonists

The main objective of this study was to investigate the ability of histamine receptor antagonists to modulate tryptase release from human colon mast cells induced by histamine. Enzymatically dispersed cells from human colon were challenged with histamine in the absence or presence of the histamine receptor antagonists, and the tryptase release was determined. It was found that histamine induced tryptase release from colon mast cells was inhibited by up to approximately 61.5% and 24% by the H1 histamine receptor antagonist terfenadine and the H2 histamine receptor antagonist cimetidine, respectively, when histamine and its antagonists were added to cells at the same time. The H3 histamine receptor antagonist clobenpropit had no effect on histamine induced tryptase release from colon mast cells at all concentrations tested. Preincubation of terfenadine, cimetidine or clobenpropit with cells for 20 minutes before challenging with histamine did not enhance the ability of these antihistamines to inhibit histamine induced tryptase release. Apart from terfenadine at 100 microg/ml, the antagonists themselves did not stimulate tryptase release from colon mast cells following both 15 minutes and 35 minutes incubation periods. It was concluded that H1 and H2 histamine receptor antagonists were able to inhibit histamine induced tryptase release from colon mast cells. This not only added some new data to our hypothesis of self-amplification mechanisms of mast cell degranulation, but also suggested that combining these two types of antihistamine drugs could be useful for the treatment of inflammatory bowel disease (IBD).     

Modulation of tryptase and histamine release from human lung mast cells by protease inhibitors

Inhibition of IgE dependent histamine release from human mast cells by protease inhibitors has been observed in skin, tonsil and synovial tissues. However, little is known about the actions of protease inhibitors on tryptase release from human lung mast cells. We therefore examined the ability of protease inhibitors to modulate tryptase and histamine release from human lung mast cells. IgE dependent tryptase release from dispersed lung mast cells was inhibited to a maximum of approximately 53.8% and 44.5% by N-a-tosyl-L-lysine chloromethyl ketone (TLCK) and N-p-Tosyl-L-phenylalanine chloromethyl ketone (TPCK), respectively. A similar degree of inhibition of calcium ionophore A23187 (CI) induced tryptase release was also observed with these two inhibitors. Preincubation of TLCK or TPCK with the mast cells at 37 degrees C for 20 minutes before addition of anti-IgE or CI did not improve their ability to inhibit anti-IgE and CI induced tryptase release. At a concentration of 10 microg/ml, protamine inhibited anti-IgE or CI induced tryptase release; but at 100 microg/ml, it increased anti-IgE and CI induced release of tryptase from lung mast cells. A concentration dependent inhibition of anti-IgE and CI induced release of histamine from lung mast cells was also observed with TLCK, TPCK and protamine. The maximum inhibition of anti-IgE induced histamine release was approximately 40.7%, 40.2% and 33.4% with TLCK, TPCK and protamine, respectively. At the concentrations tested, TLCK and TPCK by themselves did not stimulate tryptase and histamine release from lung mast cells. A specific inhibitor of aminopeptidase, amastatin, had no effect on anti-IgE induced tryptase and histamine release and was used as control. In conclusion, it was demonstrated that protease inhibitors are able to inhibit IgE dependent tryptase and histamine release from human lung mast cells, which suggested that they could be developed to a novel class of anti-inflammatory drugs to treat allergic conditions in man.     

Nitroprusside,a nitrogen oxide generating drug,inhibits release of histamine and tryptase from human skin mast cells

We have investigated the effects of the nitric oxide donor sodium nitroprusside on the immunologic release of histamine and tryptase from human mast cells. Isolated human skin mast cells were purified using Percoll gradient centrifugation. Mast cells with a purity of 70–90% were preincubated for 30 minutes with sodium nitroprusside and subsequently stimulated with anti-IgE. Upon challenge with anti-IgE, a release of 6.37±0.46 nmol histamine and 612±49 ng tryptase per 10⁶ cells was observed. In presence of sodium nitroprusside a dose-dependent decrease of the release occurred. The results suggest that nitric oxide is effective in reducing the release of histamine and tryptase from human skin mast cells.     

Selectively enhanced sensitivity of bronchoalveolar lavage fluid (BALF) mast cells to IgE dependent stimulation in mild asthmatics

The aim of this study is to investigate the histamine-releasing ability of mast cells from asthmatic bronchoalveolar lavage fluid (BALF). Following the measurement of the forced expiratory volume at the first second (FEV1), 29 mild asthmatics were included in the study and were subjected to fibreoptic bronchoscopy. The cells recovered from the BALF were challenged with anti-IgE, calcium ionophore A23187 (CI) or adenosine, and the released histamine was measured with an enzyme-linked chromogenic assay. Enzymatically dispersed mast cells from human lung or colon tissues were employed as control groups. The results showed that mast cells from BALF were at least 100 fold more sensitive to anti-IgE than those from lung or colon tissues. However, there was little difference between mast cells from BALF, lung or colon tissues in response to CI. Adenosine failed to stimulate histamine release from BALF mast cells. In conclusion, asthmatic BALF mast cells are much more sensitive to IgE-dependent stimulation than the non-IgE-dependent ones, indicating that mast cells may play a role in the pathogenesis of asthma.     

Histamine secretion from human skin slices induced by anti-IgE and artificial secretagogues and the effects of sodium cromoglycate and salbutamol

Human cutaneous mast cells show functional differences from their counterparts in other tissues. Following passive sensitization with 1% atopic serum for 30 min at 37° C human skin slices released histamine after challenge with anti-human IgE in a concentration dependent manner. Maximum release of 14 ± 2%, was achieved with a 1/10 dilution of anti-IgE. Passive sensitization with 10% atopic serum increased the secretory response to anti-IgE but histamine release was only concentration related over the entire 1/1000 to 1/10 dilution range in half of the specimens studied, the remainder showing high dose tolerance to anti-IgE, Negligible histamine release occurred with anti-IgE challenge of slices which had not been passively sensitized. The histamine releasing ability of A23187 in human skin slices was similar to that observed in lung and adenoidal mast cells being concentration dependent over the range 0-1 3 μM with a maximum release of 25 ±3%. In contrast to human lung and adenoidal mast cells, poly-L-lysine and compound 48/80 induced histamine release from skin slices. Poly-L-lysine induced a concentration-dependent release of histamine over the range 0-01-10 mUM with a maximum of 27 ± 3%. The response to compound 48/80 was variable, releasing in some but not all specimens. Histamine release caused by anti-IgE. A23187 and poly-L-lysine was shown to be dependent upon extracellular calcium while release stimulated by compound 48/80 was calcium independent. The chemotactic peptide, formyl-methionyl-leucyl-phenylalanine, over the range 0.01 10 μM failed to release histamine from skin slices. Sodium cromoglycate (100 1000 μM) failed to inhibit histamine release and the β-adrenoceptor stimulant salbutamol (1-10 μM) showed only weak activity at the lowest of three different concentrations of anti-IgE used for challenge.     

腺苷和抗IgE抗体对哮喘患者BALF中肥大细胞分泌组胺的影响

目的 :研究支气管哮喘 (哮喘 )患者支气管肺泡灌洗液 (BALF)中肥大细胞的功能特性。方法 :2 9例轻度哮喘患者BALF中的细胞经冲洗后 ,加入含抗IgE抗体、腺苷、缓冲液或腺苷 +抗IgE抗体的LP4试管中进行激发试验。检测BALF肥大细胞中的组胺释放量。结果 :腺苷浓度达 10 0 μmol/L时 ,仍无明显刺激组胺释放的作用 ,仅在浓度高达 10 0 0 μmol/L时 ,方可诱导哮喘患者BALF中肥大细胞释放约 17%的组胺。抗IgE抗体的浓度大于 1× 10 -4g/L时对哮喘患者BALF中肥大细胞的释放组胺有明显的促进作用 ;仅 3× 10 -5g/L的抗IgE抗体与腺苷联合应用的实测值 ,明显高于对应的单独作用组的相加值。结论 :哮喘患者BALF中的肥大细胞对腺苷的刺激反应较差 ,但对抗IgE抗体刺激的反应性明显增强。仅低浓度的抗IgE抗体和腺苷具有协同作用。