EXOCYTOSIS & RECYCLING IN RBL 2H3 MUCOSAL MAST CELLS

胞吐作用

• 批准号: 6349382
• 负责人: REBECCA M WILLIAMS
• 金额: $ 6.18万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-01 至 2001-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6349382
• 关键词: Mammalia; animal tissue; bioengineering /biomedical engineering; biological products; biomedical resource; hematology; human tissue; immunology; inflammation; lasers; lipids; lymphatic system

The mucosal mast cell line RBL-2H3 secrete histamine, serotonin (5-hydroxytryptamine, 5-HT) and other inflammatory mediators in response to applied allergen. Previously it has been shown that 5-HT distributions can be imaged in 5-HT loaded RBL-2H3 cells using three photon excitation (3PE) of 5-HT autofluorescence (Maiti et al., 1997, Science 275:530). In this application of multiphoton microscopy, an infrared (740 nm), ultrafast (pulsed) laser was used to excite the < 300 nm 5-HT electronic transition, alleviating chromatic aberrations and the severe photodamage associated with deep UV wavelengths. We are making 3PE imaging measurements of RBL-2H3 cell secretion. During imaging the cells are observed to characteristically ruffle and spread as granular 5-HT autofluorescence disappears with no detectable granule movement. Individual cells exhibit highly heterogeneous release kinetics, often with quasi periodic bursts. Neighboring granule disappearances are generally correlated, indic ative of either spatially localized signaling or granule-granule interactions. After individual granule release events, weak residual fluorescence patterns retaining the initial granule shapes are often visible, suggesting leftover 5-HT still bound to the granule matrix. In two-color experiments, we are using a LysoSensor dye to simultaneously record the pH history of granules as they secrete. Using this dye pH jumps are observed and we are presently examining their temporal relation to calcium oscillations that also occur upon cell stimulation.

粘膜肥大细胞系 RBL-2H3 分泌组胺、血清素 （5-羟色胺，5-HT）和其他炎症介质 对所施加的过敏原的反应。 此前已有研究表明，5-HT 可以使用三种方法在加载 5-HT 的 RBL-2H3 细胞中对分布进行成像 5-HT 自发荧光的光子激发 (3PE)（Maiti 等人，1997， 科学 275:530）。 在多光子显微镜的应用中， 红外（740 nm）、超快（脉冲）激光用于激发< 300 nm 5-HT电子跃迁，减轻色差 以及与深紫外线波长相关的严重光损伤。 我们 正在对 RBL-2H3 细胞分泌进行 3PE 成像测量。 期间 成像时观察到细胞具有典型的褶皱和扩散特征 随着颗粒状 5-HT 自发荧光消失，无法检测到 颗粒运动。 单个细胞表现出高度异质性 释放动力学，通常具有准周期性爆发。 邻接 颗粒消失通常是相关的，表明 空间局部信号传导或颗粒间相互作用。 后 单个颗粒释放事件，微弱的残留荧光模式 保留初始颗粒形状通常是可见的，这表明 剩余的 5-HT 仍与颗粒基质结合。 两种颜色的 实验中，我们使用 LysoSensor 染料同时记录 颗粒分泌时的 pH 历史。 使用这种染料 pH 值会跳跃 被观察到，我们目前正在检查它们与 细胞刺激时也会发生钙振荡。