【摘要】 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。更多还原

【Abstract】 AIM: To investigate the ability of Chinese cobra snake venom-metalloproteinase（MT） to induce the histamine release from human mast cells and its potential mechanisms.METHODS: MT was purified from the snake venom by using heparin agarose and Superdex75 chromatography.Mast cells were dispersed from human lung, colon and tonsil tissues after digestion with collagenase and hyaluronidase.The dispersed mast cells were then challenged with MT,stimulus and control in LP4 tubes for 15 min at 37 ℃.A glass fibre-based fluorometric assay was used to measure histamine in the supernatants of dispersed mast cells.RESULTS: MT induced a dose-dependent release of histamine from human colon,lung and tonsil mast cells.As low as 0.03（mg/L） of MT was able to stimulate significant histamine release from human colon mast cells,but a minimum of 0.3 or 30 mg/L of MT was required to stimulate a similar level of histamine release from lung or tonsil mast cells,respectively.The release of histamine from colon and lung mast cells in response to MT was maximized at 12 min following the addition of the stimulus.This was quite different from the picture of the peak histamine release from tonsil mast cells,in which histamine release was maximized at 8 min following the addition of MT.Pretreatment of cells with metabolic inhibitors and pertussis toxin reduced dramatically histamine release from human colon,lung and tonsil mast cells by MT.In exogenous Ca²⁺ and Mg²⁺ free experiments,the release of histamine induced by MT was significantly decreased.CONCLUSION: Cobra snake venom MT induces human mast cells to release histamine through a G-protein-related mechanism,which may contribute to the pathogenesis of venomous snake bite.更多还原