Development of an Analyzing System of the Cellular Responses to Multiple Stresses

近年来，从新药开发和临床分析的角度认识到微生物检测对活细胞应激信号的重要性。本研究旨在建立细胞对多种应激信号反应的分析系统。多通道亚微米毛细管（MMC）用于向特定植物细胞施加化学或电胁迫信号，同时检测该细胞或另一个细胞的响应信号。反应信号是化学的、电的或电化学的。将阴离子荧光染料lucifer yellow （LY）引入培养烟草（BY-2，衍生自Nicotiana tabacum l.v . bright yellow -2）或弗吉尼亚烟（Tradescantia virginia l.l .）的叶细胞中作为化学信号。发现LY的扩散模式是确定毛细管尖端位置的有用指标。同样，将H^+、K^+、Cl注入细胞中，用MMC设计的离子选择微电极确定其注入。在细胞内注射荧光素酶和脲酶等酶的反应也在体内进行了测定。为了准确估计作用于细胞膜之间的电压，采用膜片箝位放大器和锁相放大器设计了一种高性能阻抗分析系统。例如，测定BY-2细胞间膜的膜阻抗为2-7G ω和500pF的并联电路。本系统能够分别或同时或重复地对同一单个细胞施加多个应力信号。这是研究应力信号强度与细胞响应强度之间复杂关系的一个显著优势。

Microbioassay for stress signal to living cells has recently been recognized important from the viewpoint new drug development and clinical analysis. This study aimed to develop an analyzing system of cellular responses to multiple stress signals. Multi-channnel sub-micron capillary (MMC) was used to apply a chemical or electrical stress signal to a particular plant cell and, at the same time, to detect the response signal of the cell or another cell. The response signals were chemicel, electrical, or electrochemical.Anionic fluorescence dye, lucifer yellow (LY), was introduced into a cell of cultured tobacco (BY-2, derived from Nicotiana tabacum L.cv.Bright Yellow-2) or a leaf cell of Tradescantia virginiana L.as an example of a chemical signal. The diffusion pattern of LY was found a useful indicator to confirm the tip position of the capillary. In the same way, H^+, K^+, Cl were injected in a cell and its injection was ascertained with respective ion-selective microelectrodes which were devised with MMC.The reaction of enzymes such as luciferase and urease which were injected in a cell was also measured in vivo.In order to estimate an exact voltage acting between the intercellular membrane, a high performence impedance analyzing system was developed using a patch-clamp amplifier and a lock-in amplifier. The membrane impedance of a intercellular membrane of BY-2 was, for example, determined as a parallel circuit of 2-7G OMEGA and 500pF.The present system has enabled the application of multiple stress signals separately or simultaneously, or repeatedly to the same single cell. This is a marked advantageous point in studying complicated relationships between stress signal intensity and cellilar response intensity.