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Development of a system for the continuous and simultaneous measurement of vascular intracellular signalings and metabolism

开发连续同步测量血管细胞内信号传导和代谢的系统

基本信息

批准号：
13557067
负责人：
KANAIDE Hideo
金额：
$ 7.81万
依托单位：
Kyushu University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

项目摘要

The only successful technique for "the continuous and simultaneous monitoring of the intracellular signalings, function and metabolism of blood vessels, without destructing the tissue structure" must be the front-surface fluorimetry. In the first year of this study, we have newly developed a system to carry out 3 sets of dual-wavelength front-surface fluorimetries simultaneously. Followings are characteristic features of the system: (1) In collaboration with the Japan Spectroscopic Co. (JASCO, Tokyo, Japan), we have developed a "shuttering system" which controls the on/off of the excitation lights from 3 sets of fluorimeters( CAM-OF), When 1 fluorimeter is on, the other 2 are off. Thus, it is possible to obtain the information of emission of every fluorimeter for 1s every 3s, for 2s every 6s, for 4s every 12s, for 8s every 24s, or for 16s every 48s. (2) In collaboration with FUJITOK Co. (Tokyo, Japan), we have specifically designed and made the fiber-optic light guide for front-surface fluorimetry. The light guide utilizes 3 sets of quartz fibers and 3 sets of glass fibers, which are connected to 3 light sources and 3 photomultipliers at one end," respectively. Fibers are concentrically arranged at the common end, which faces to the samples. The quartz fibers are arranged in an inner circle, whereas the glass fibers are arranged in an outer circle. We also made a light guide with fibers arranged at random at the common end. Using this system, we tried to measure [Ca^<2+>]I (fura-2; Em: 500nm, Ex: 340/380nm) and (pH)I (BCECF; Em; 540nm, Ex: 500/450nm), simultaneously, and found out that 500nm Em-light for BCECF interfered the 500nm Ex-light for fura-2. In the second year, we have modified the "shuttering system", which will solve this problem. Using this system, we have successfully carried several studies about the Ca^<2+> signalings in the vascular smooth muslcs.

“在不破坏组织结构的情况下，连续和同时监测血管的细胞内信号、功能和代谢”的唯一成功的技术必须是前表面荧光法。在本研究的第一年，我们新开发了一个系统，同时进行3套双波长前表面荧光分析。（1）与日本分光公司（JASCO，Tokyo，Japan）合作，开发了一种“快门系统”，它控制来自3套荧光计（CAM-OF）的激发光的开/关，当1台荧光计打开时，另外2台荧光计关闭。因此，可以每3s获得1 s，每6s获得2s，每12 s获得4s，每24 s获得8 s，或每48 s获得16 s。(2)我们与FUJITOK Co.（日本东京）合作，专门设计并制造了用于前表面荧光测定的光纤光导。光导利用3组石英纤维和3组玻璃纤维，它们在一端分别连接到3个光源和3个光电倍增管。纤维同心地布置在面向样品的公共端。石英纤维布置在内圆中，而玻璃纤维布置在外圆中。我们还制作了一个光导，其纤维在公共端随机排列。利用该系统，我们尝试同时测定[Ca^<2+>]I（Fura-2; Em：500 nm，Ex：340/380 nm）和（pH）I（BCECF; Em：540 nm，Ex：500/450 nm），发现BCECF的500 nm EM光对Fura-2的500 nm Ex光有干扰。第二年，我们修改了“模板系统”，解决了这个问题。利用该系统，我们已经成功地进行了几项关于血管平滑肌Ca^2+信号转导的研究。