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

最近，从新药开发和临床分析的角度来看，对活细胞的应激信号的微生物学分析已经被认为是重要的。本研究旨在开发细胞对多种应激信号的响应分析系统。多通道亚微米毛细管(MMC)被用来向特定的植物细胞施加化学或电应力信号，同时检测该细胞或另一细胞的响应信号。将阴离子荧光染料荧光黄(LY)导入烟草栽培细胞(BY-2，来源于烟草BY-2)或紫露草叶片细胞中，作为化学信号的例子。LY的扩散模式是确定毛细管尖端位置的一个有用的指示剂。以同样的方法将H^+、K^+、Cl注入细胞内，并用MMC设计相应的离子选择性微电极来确定其注入。同时还活体测量了注入细胞内的荧光素酶和尿素酶等酶的反应。为了准确地估计细胞间膜间的电压，利用膜片钳放大器和锁定放大器研制了一种高性能的阻抗分析系统。例如，BY-2的细胞间膜的膜阻抗被测定为2-7G欧米伽和500pF的并行电路。本系统能够单独或同时或重复地将多个应力信号施加到同一单个细胞。这对于研究胁迫信号强度和细胞反应强度之间的复杂关系是一个明显的优势。

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.