Bio-Cell Gene Expression Analyzer

“生物细胞”一词是指在整个施加压力的实验过程中保持其生存状态的细胞。本研究是为了实时分析生物细胞对各种应激信号的反应。对培养的烟草或水稻细胞施加电胁迫信号。作为细胞反应，用显微荧光图像分析系统检测基因表达。以烟草细胞By -2系为实验材料，测量了在不同强度的脉冲电胁迫信号作用下细胞膜的电阻抗。结果表明，当跨膜电位（V_<CMP>）超过200mV时，可观察到明显的膜透性。另一方面，利用水稻原生质体两步实验证实了几丁质酶基因的Ca^<2+>依赖性表达。第一步，通过脉冲电应力信号控制Ca^<2+>的引入。结果，Ca^<2+>在VCMP的1-5 mV左右进入细胞。第二步，我们通过将Ca^<2+>直接注入原生质体，证实了几丁质酶基因的表达。除了电应力外，我们还打算在生物电池的内部施加电化学应力。然后我们制作了直径为1-2 μm的毛细管碳微电极，并通过分析多巴胺的环伏安图来检验其性能。综上所述，建立了生物细胞基因表达分析系统，并展示了其良好的应用前景。

The term "bio-cell" means a cell that maintains its viable state throughout the experiments of stress application. This study was done in order to demonstrate the real time analysis of the bio-cell responses to various stress signals. An electric stress signal was applied to cultured cells of tobacco or rice. As the cell response, the gene expression was measured with a microscopic fluorescent image analyzing system. By the experiment using tobacco cells, line BY-2, the electric impedance of the cell membrane was measured under various intensities of pulsing electric stress signals. As the result, appreciable membrane permeabilization was observed when the cross membrane potential (V_<CMP>) exceeded 200mV.On the other hand, the Ca^<2+>-dependent expression of chitinase gene was demonstrated by 2-step experiments using rice protoplasts. At the first step, Ca^<2+> introduction was controlled by the pulsing electric stress signal. As the result, Ca^<2+> entered the cells around 1-5 mV of VCMP.As the 2nd step, we demonstrated the chitinase gene expression by the direct injection of Ca^<2+> into the protoplasts. Besides the electric stress, we intended to apply an electrochemical stress to a bio-cell at its inside. Then we developed a capillary carbon microelectrode with a 1-2 μm diameter and checked its performance by analyzing cyclicvoltammograms of dopamine. In conclusion, the analyzing system of the gene expression of a bio-cell was developed and its promising performance was demonstrated.