Ca2+ induced surfactant secretion in alveolar type II cultures isolated from the H-2Kb-tsA58 transgenic mouse
Ca2+ induced surfactant secretion in alveolar type II cultures isolated from the H-2Kb-tsA58 transgenic mouse
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DOI：
10.1159/000083648
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发表时间：
2005-01-01
影响因子：
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通讯作者：
Dietl, P
中科院分区：
文献类型：
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作者：
Jennings, P;Bertocchi, C;Dietl, P
背景/目的：转基因小鼠的发展需要阐明表面活性物质分泌的分子机制。然而，目前对小鼠肺泡II型(AT II)细胞表面活性物质胞吐的调控知之甚少。方法：将分离自永生化小鼠的AT-II细胞在33℃培养，在干扰素存在的情况下，产生永生的小鼠AT-II细胞(IMAT-II)。用实时荧光成像法测定肺表面活性物质的分泌。结果：大鼠AT-II细胞内板层小体相关囊泡的特异性染料LTG染色显示IMAT-II细胞。透射电子显微镜证实，LBS的特征是表达密集的层状结构。用脂类染色染料FM1-43分析，笼状钙离子的闪光光解能特异性地提高细胞内钙离子浓度([Ca2+](I))，导致LB与质膜融合。用ATP(10微米)刺激嘌呤能也能引起细胞内[Ca~(2+)](I)升高(用Fura-2测定)，然后进行Lb融合。结论：iMATII细胞在多代传代中维持LBS的表达。这些细胞的表面活性物质的分泌受[Ca~(2+)](I)的调节，表现出与大鼠AT-II细胞相似的特征。这些细胞将有助于研究基因修饰对调节表面活性物质分泌的影响。版权所有(C)2005 S.Karger AG，巴塞尔。
Background/ Aims: There is a need for the development of transgenic mice to elucidate molecular mechanisms in surfactant secretion. However at present very little is known about the regulation of surfactant exocytosis in murine alveolar type II ( AT II) cells. Methods: We brought AT II cells isolated from the Immorto mouse into culture at 33degreesC, in the presence of interferon, to generate immortal mouse AT II cells ( iMAT II). Surfactant secretion was measured using real- time fluorescence imaging. Results: iMAT II cells stained with lysotracker green ( LTG), a dye specific for lamellar body related vesicles in rat AT II cells. Expression of densely layered structures, characteristic of LBs, was confirmed by transmission electron microscopy. Flash photolysis of caged Ca2+, which specifically elevates intracellular Ca2+ concentration ([ Ca2+] (i)), resulted in LB fusion to the plasma membrane, as analysed using the lipid staining dye FM 1- 43. Purinergic stimulation with ATP ( 10 muM), also resulted in a rise in [ Ca2+] (i) ( measured by fura- 2), which was followed by LB fusion. Conclusions: iMATII cells maintain the expression of LBs over several passages. Surfactant secretion in these cells is regulated by [ Ca2+] (i), and exhibits similar characteristics to that of rat AT II cells. These cells will be beneficial in studying the impact of genetic modifications on regulated surfactant secretion. Copyright (C) 2005 S. Karger AG, Basel.