ROLE OF INTRACELLULAR FREE CALCIUM IN CELL PROLIFERATION

细胞内游离钙在细胞增殖中的作用

• 批准号: 3278937
• 负责人: ROGER Y TSIEN
• 金额: $ 11.11万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-09-01 至 1991-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3278937
• 关键词: B lymphocyte; T lymphocyte; calcium; cell cell interaction; cell growth regulation; cell population study; fibroblasts; flow cytometry; fluorescence microscopy; ionophores; laboratory mouse; leukocyte activation /transformation; lymphokines; membrane proteins; mitogens; neoplastic growth; spectrometry; videotape /videodisc; viral leukemogenesis

The biological focus is to study the roles of cytosolic free Ca2+ ([Ca2+]i) changes in helping trigger the entry of quiescent cells into the cell cycle and in controlling their progression through mitosis. Emphasis will be placed on resolving differences in [Ca2+]i between heterogeneous neighboring cells and between different regions of large mitotic and motile cells. Methodology developed during the previous grant period offers the unprecedented sensitivity and spatial resolution needed. New analogs of the tetracarboxylate Ca2+ indicator quin2 offer about thirty-fold increased brightness of fluorescence, with change in excitation and sometimes emission wavelengths in response to Ca2+. Wavelength shifts mean that [Ca2+]i changes can now be detected by ratios of fluorescence at two wavelengths, without interference from variations in dye content or cell size, in individual 6 um cells studied by fluorescence microscopy or by flow cytrometry. Spatial resolution to image [Ca2+]i merely requires a video processing system whose feasibility has been demonstrated by collaboration with a better-equipped laboratory. This system will be used to examine the time course of [Ca2+]i in individual lymphocytes treated with mitogenic and some non-mitogenic lectins, antibodies, and lymphokines. Lymphocyte surface markers will be assessed by the usual antibodies to selectively stain cells or adhere them to the chamber floor. Flow cytometry will provide an independent view of the population statistics. The video system will also be used to study [Ca2+]i and pHi in fibroblasts stimulated with growth factors or transformed by a temperature-sensitive oncogene. Present studies showing [Ca2+]i rises correlated with several major events in mitosis of sea urchin zygotes will be extended by the imaging capability to see if and where the rises are localized in the cell. Cell lines such as PtK1 or CHO will be checked to see if they too have [Ca2+]i fluctuations. The importance of [Ca2+]i transients during mitosis should be tested by using photoreactive Ca2+ chelators to generate spatially and temporally defined rises or falls in [Ca2+]i. Meanwhile, chemical efforts will be devoted to yet further improvements of [Ca2+]i indicators by increasing their wavelengths of operation. Further work on photoreactive Ca2+ chelators is aimed at increasing the quantum efficiency, speed, and wavelengths of photolysis. Prototype Na+ selective indicators need optimization of fluorescent properties and addition of carboxylate groups to make them physiologically useful.

细胞内游离钙离子（[Ca 2 +]i）的作用是生物学研究的重点。 帮助触发静止细胞进入细胞周期的变化 并通过有丝分裂控制它们的进程。 重点将 用于解决异质性细胞间[Ca2+]i的差异 相邻的细胞和大的有丝分裂和运动的不同区域之间 细胞 在上一个赠款期间制定的方法提供了 需要前所未有的灵敏度和空间分辨率。 新的类似物 四羧酸盐Ca2+指示剂quin2提供约30倍增加 荧光亮度，随着激发的变化，有时 响应于Ca2+的发射波长。 波长偏移意味着 [Ca2+]i的变化现在可以通过在两个 波长，不受染料含量或细胞变化的干扰 大小，在通过荧光显微镜或 流式细胞术 图像[Ca2+]i的空间分辨率仅需要 视频处理系统，其可行性已被证明， 与设备更好的实验室合作。 该系统将用于 检查处理的单个淋巴细胞中[Ca2+]i的时间过程 与促有丝分裂和一些非促有丝分裂凝集素、抗体和淋巴因子。 淋巴细胞表面标志物将通过常规抗体进行评估， 选择性地染色细胞或将它们粘附到室底。 流 细胞计数将提供一个独立的人口统计数据。 该视频系统还将用于研究成纤维细胞中的[Ca 2 +]i和pHi 用生长因子刺激或用温度敏感的 癌基因 目前的研究表明，[Ca2+]i升高与几种 海胆受精卵有丝分裂的主要事件将被延长， 成像能力，以查看是否以及在哪里的崛起是局部的， cell. 将检查PtK1或CHO等细胞系，以确定它们是否也 有[Ca2+]i波动。 [Ca2+]i瞬变的重要性 有丝分裂应通过使用光反应性Ca2+螯合剂来测试， [Ca2+]i在空间和时间上定义的上升或福尔斯。 与此同时，化学方面的努力将致力于进一步改善 [Ca2+]i指示器通过增加它们的操作波长。 进一步 光反应性Ca2+螯合剂的工作旨在增加量子 效率、速度和光解波长。 原型Na+选择性 指示剂需要优化荧光特性， 羧酸酯基团，使其在生理上有用。