EGTA treatment of human airways in vitro unmasks M1/MUC5AC mucin in submucosal glands.
EGTA treatment of human airways in vitro unmasks M1/MUC5AC mucin in submucosal glands.
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DOI：
10.1183/09031936.01.00105301
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发表时间：
2001-07
期刊：
影响因子：
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通讯作者：
Patricia Roger;J. Gascard;J. Bara;É. Dulmet;C. Brink
中科院分区：
文献类型：
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作者：
Patricia Roger;J. Gascard;J. Bara;É. Dulmet;C. Brink
粘蛋白染色可用于评估人类气道的分泌活性。然而，粘蛋白表位可能被分泌物的理化特性所掩盖。本研究的目的是检查钙螯合剂乙二醇-双-(β-氨基乙基醚)-N,N,N',N'-四乙酸 (EGTA) 对分离的人支气管制剂中 M1/MUC5AC 粘蛋白检测的影响。使用抗 M1 单克隆抗体 (Mab) 进行免疫组织化学研究和免疫放射测定，检测源自具有完整表面上皮的支气管制剂或上皮已去除的组织（擦拭制剂）中的 M1/MUC5AC 粘蛋白。 Mab 标记了暴露于 EGTA (4 mM) 的支气管制剂中的上皮杯状细胞和粘膜下腺细胞，而在暴露于 EGTA (0.4 mM) 的组织中仅杯状细胞被染色。与未处理的对照值相比，在源自 EGTA (4 mM) 暴露的完整和摩擦制剂的支气管液中或在用 EGTA (4 mM) 处理的支气管液中检测到的 M1/MUC5AC 粘蛋白量显着增加两倍 (p<0.001)。此外，在人体气道暴露于 EGTA (4 mM) 期间，乳酸脱氢酶 (LDH) 活性和蛋白质测量值未发生改变，表明这种处理不会影响组织活力。这些结果证明乙二醇-双-(β-氨基乙基醚)-N,N,N',N'-四乙酸 (4 mM) 通过改变呼吸道粘蛋白的理化性质，从而暴露与抗 M1 单克隆抗体发生反应的表位，从而促进 M1/MUC5AC 粘蛋白的检测。这将能够更准确地测量体外人体气道的分泌活动。
Mucin staining can be used to evaluate secretory activity of human airways. However, mucin epitopes may be masked by physicochemical properties of the secretions. The aim of this investigation was to examine the effects of the calcium chelator, ethyleneglycol-bis-(beta-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) on the detection of M1/MUC5AC mucin in isolated human bronchial preparations. Immunohistochemical investigation and immunoradiometric assays with anti-M1 monoclonal antibodies (Mabs) were used to detect M1/MUC5AC mucin derived from bronchial preparations with an intact surface epithelium, or in tissues where the epithelium had been removed (rubbed preparations). The Mabs labelled both epithelial goblet cells and submucosal glandular cells in EGTA (4 mM)-exposed bronchial preparations, while only goblet cells were stained in EGTA (0.4 mM)-exposed tissues. The quantities of M1/MUC5AC mucin detected in either the bronchial fluids derived from EGTA (4 mM)-exposed intact and rubbed preparations or in bronchial fluids treated with EGTA (4 mM) were significantly increased by two-fold when compared with untreated control values (p<0.001). In addition, lactate dehydrogenase (LDH) activity and protein measurements were unaltered during exposure of human airways to EGTA (4 mM) suggesting that this treatment did not affect tissue viability. These results provide evidence that ethyleneglycol-bis-(beta-aminoethylether)-N,N,N',N'-tetraacetic acid (4 mM) facilitates the detection of M1/MUC5AC mucin by altering the physicochemical properties of respiratory mucin, thereby exposing epitopes with which anti-M1 monoclonal antibodies are reactive. This will allow more accurate measurement of secretory activity in human airways in vitro.