Development of in vitro reconstituted system for investigating intracellular signal trasduction and cellular function in myocardial cells

本研究的目的是开发一种体外实时分析系统，用于CCD相机研究细胞内信号转导。1995年，我们成功地开发了一种体外重建系统，用于分析细胞内信号转导，特别是核信号转导。研究发现，核蛋白在30分钟内被转运到细胞核中，而非核蛋白则不能通过心脏匀浆转运到细胞核中，这意味着该系统可以复制体内核蛋白转运。1996年，我们用这种体外系统检测了患病心脏提取物的核转运效率。自发性高血压大鼠的核转运率明显低于对照组。此外，我们发现了被核定位信号（NLS）激活的细胞因子的存在，并对这些细胞因子进行了部分纯化（FEBS Letters）。下一步，我们需要对该系统进行改进，以便用CCD相机定量分析分子的实时行为。实时分析系统将提供核转运效率的精确评估，可能调节心脏细胞的细胞功能。

The aim of this research is to develop an in vitro real-time analyzing system for investigating intracellular signal transduction with CCD camera. In 1995, we succeeded to develop an in vitro reconstituted system for analyzing of intracellular signal transduction, especially nuclear signal transduction. It was found that nuclear protein was transported to the nucleus within 30 minutes, although non-nuclear proteins were not by using homogenates from heart which means that this system reproduces in vivo nuclear protein transport. In 1996, we examined nuclear transport efficiency with extracts from diseased hearts by this in vitro system. The rate of the nuclear transport was more decreased in spontaneous hypertensive rat than of the control. Furthermore, we found the existence of cellular factors that are activated by nuclear localization signal (NLS) and partially purified these cellular factors (FEBS Letters). In the next step, we should improve this system in order to analyze the real-time behavior of molecules quantitatively with CCD camera. The real-time analytical system will provide the precise evaluation of efficiency of nuclear transport that may modulate the cellular function in heart cells.