Nuclear Calcium Signals in Immune Responses

免疫反应中的核钙信号

基本信息

批准号：
04454621
负责人：
NAKANISHI Mamoru
金额：
$ 3.84万
依托单位：
Nagoya City University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1993
项目状态：
已结题

项目摘要

We have studied receptor-mediated calcium signals in antigen-specific B cells (trinitrophenol-specific B cell clone, TP67.21) and rat basophilic leukemia cells (RBL-2H3) using a confocal fluorescence microscope with an argon ion laser (488nm) and a He-Cd laser (325nm). Confocal fluorescence images of fluo-3-loaded B cells and RBL-2H3 cells, excited by an argon ion laser, became much brighter and more nonhomogeneous than those before antigen stimulation. Time-dependent fluorescence changes in intensities were abrupt and quite similar to the patterns of the intracellular calcium ion concentration [Ca^<2+>]_i observed by a conventional fluorescence microscope using fura-2. From the morphological patterns of the calcium images, the parts of the bright fluorescence seemed to belong to the nucleus in B cells (or RBL-2H3 cells). To confirm the above events we measured the confocal fluorescence images of the nucleus. From the fluorescence images of co-loaded Hoechst 33342 (a DNA-specific fluorescence probe), which excited by a He-Cd laser, the brighter parts of the fluo-3 fluorescence intensities were identified to the nucleus in B cells and RBL-2H3 cells. These nuclear calcium signals were also observed by the addition of thapsigargin, an inhibitor of the intracellular calcium pump. These results suggested the possibility that the increased intranuclear calcium ions may play a nuclear third messenger in B cells and RBL-2H3 cells. Then, we tried to determine whether the increased intranuclear calcium ions are transferred from the cytoplasm or they are released from the nuclear stores. We used Ba^<2+> and Mn^<2+> instead of Ca^<2+>, because Ba^<2+> and Mn^<2+> are known to enter via Ca^<2+> channels but they quench the fluo-3 fluorescence. The results showed that Ba^<2+> and Mn^<2+> entered into the nucleus as well as into the cytoplasm and quenched the fluo-3 fluorescence both in the nucleus and in the cytoplasm. This sug

我们使用带有氩离子激光（488nm）的共聚焦荧光显微镜，研究了抗原特异性 B 细胞（三硝基苯酚特异性 B 细胞克隆，TP67.21）和大鼠嗜碱性白血病细胞（RBL-2H3）中受体介导的钙信号， He-Cd 激光器 (325nm)。负载fluo-3的B细胞和RBL-2H3细胞的共焦荧光图像在氩离子激光激发下变得比抗原刺激前更亮、更不均匀。随时间变化的荧光强度变化是突然的，并且与使用fura-2的传统荧光显微镜观察到的细胞内钙离子浓度[Ca 2+ ]_i 的模式非常相似。从钙图像的形态图案来看，明亮荧光的部分似乎属于B细胞（或RBL-2H3细胞）的细胞核。为了证实上述事件，我们测量了细胞核的共焦荧光图像。从共同加载的 Hoechst 33342（一种 DNA 特异性荧光探针）的荧光图像中，该探针由 He-Cd 激光激发，fluo-3 荧光强度的较亮部分被识别到 B 细胞和 RBL-2H3 的细胞核细胞。通过添加毒胡萝卜素（一种细胞内钙泵抑制剂）也可以观察到这些核钙信号。这些结果表明，增加的核内钙离子可能在 B 细胞和 RBL-2H3 细胞中发挥核第三信使的作用。然后，我们试图确定增加的核内钙离子是从细胞质转移还是从核储备中释放。我们使用 Ba^<2+> 和 Mn^<2+> 代替 Ca^<2+>，因为已知 Ba^<2+> 和 Mn^<2+> 通过 Ca^<2+> 通道进入但它们会淬灭 Fluo-3 荧光。结果表明，Ba^2+和Mn^2+不仅进入细胞核，而且进入细胞质，猝灭细胞核和细胞质中的fluo-3荧光。这个建议