活细胞实时动态观测与量化分析及应用
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摘要
活细胞是生命体基本结构单位和功能单位，因此对单个活细胞的实时荧光观测对生命科学有重要意义，而深入量化分析实验数据有助于揭示生命科学中的信息。本论文改进了多功能高灵敏实时荧光显微成像系统及其图像处理方法，使其适合活细胞长时间实时荧光成像，并将该系统应用于小鼠胸腺细胞凋亡早期生物事件研究和中药抑制海马神经元网络自发同步钙振荡研究中。
首先，设计搭建活细胞培养小室，改进多功能高灵敏实时荧光显微成像系统相关图像处理方法，使系统在实现实时荧光观测、全内反射荧光成像、双通道同时成像基础上更适合活细胞长时间实时荧光观测和数据分析。
其次，在亚硝基谷胱甘肽（GSNO）诱导小鼠胸腺细胞凋亡早期研究中，不仅观测到GSNO作用胸腺细胞后胞内游离钙离子浓度（[Ca2+]i）迅速增加并达到一个平台、线粒体跨膜电位（ΔΨm）下降、细胞内溶酶体pH降低及凋亡细胞胞质酸化。还使用量化分析方法分析大量单个细胞实验数据，计算拟合曲线拐点T和半最大值t1/2两个特征参数。发现[Ca2+]i、ΔΨm、溶酶体pH曲线的拐点（分别记为TCa、Tmito和Ti）数值上满足TCa 最后，研究了不同中药配伍对海马神经元网络自发同步钙振荡的抑制作用，观测了中药五味子甲素、乙素不同配伍对海马神经元网络自发同步钙振荡抑制情况和胡椒碱、甲素不同配伍对自发同步钙振荡抑制情况。通过对抑制频率和抑制振幅的量化分析发现，不同配伍对频率和振幅抑制作用不同，并且对频率抑制作用与对振幅抑制作用不相关。根据神经细胞钙离子浓度变化来源建立神经细胞钙离子振荡动力学模型，并对模型中7个参数对钙振荡频率和振幅的影响进行了分析。
本文的研究将荧光显微成像技术应用于具体活细胞荧光观测和量化分析中，并将为活细胞生命科学提供有利的技术和方法。
Living cells are the basic structural units and functional units of organisms.Therefore, real-time dynamic observation in single living cells is of great importance inlife sciences and quantitative analysis the experiment data in depth is helpful to revealthe information of life sciences. In this thesis, a real-time multifunctional fluorescencemicroscopic imaging system as well as relevant image processing methods is improvedfor long term living cells observation. The studies of early apoptosis on thymocytes andthe spontaneous and synchronous intracellular Ca2+oscillations of cultured hippocampalneuronal networks are carried out in the system.
First, the real-time multifunctional fluorescence microscopic imaging systembecame more suitable for long term living cells observation after the installation ofincubator and the development of relevant image processing methods.
Secondly, a series of changes in thymocytes early apoptosis induced byS-nitrosoglutathione (GSNO) were studied. During the early apoptosis, the increase ofintracellular free calcium ion concentration ([Ca2+]i), the loss of mitochondrialmembrane potential (ΔΨm), the decrease of lysosomal pH, cytoplasmic acidification ofapoptosis cells were detected. The kinetic features were quantitatively analyzed andcompared by fitting the experiment data. The mathematical parameters, inflection pointand half-max effect point were proposed to analyze the fitting curve. The resultsrevealed that the inflection point of lysosomal pH always appeared prior to that of ΔΨmand posterior to that of [Ca2+]iin a certain GSNO concentration induced. Half-maxeffect point was also displayed the similar results. Such quantitative analyses ofreal-time observations are useful for interpreting the sequence of the biological eventsoperating in GSNO-induced thymocyte apoptosis and provide important clues forapoptotic signaling pathway studies.
Finally, the modulation of different compatibilities of Chinese herbal medicine onsynchronized Ca2+oscillations in cultured hippocampal neuronal networks were carriedout. We investigated and quantitative analyzed the modulation effects of lowconcentration and different compatibilities on synchronized Ca2+oscillations of piperineand deoxyschizandrin, deoxyschizandrin and schisandrin B in cultured hippocampalneuronal networks. The results suggest that different compatibilities have different inhibition on the synchronized Ca2+oscillations. The inhibitory effect on the frequencyis irrelevantly to that on the amplitude. A calcium oscillation model of nerve cells wassetup and the effects of its seven parameters were analyzed.
This thesis is a specific application of fluorescence microscopic imagingtechniques in living cells and quantitatively analyses and supposed to provideadvantageous techniques and methods to studies on living cells.
首先，设计搭建活细胞培养小室，改进多功能高灵敏实时荧光显微成像系统相关图像处理方法，使系统在实现实时荧光观测、全内反射荧光成像、双通道同时成像基础上更适合活细胞长时间实时荧光观测和数据分析。
其次，在亚硝基谷胱甘肽（GSNO）诱导小鼠胸腺细胞凋亡早期研究中，不仅观测到GSNO作用胸腺细胞后胞内游离钙离子浓度（[Ca2+]i）迅速增加并达到一个平台、线粒体跨膜电位（ΔΨm）下降、细胞内溶酶体pH降低及凋亡细胞胞质酸化。还使用量化分析方法分析大量单个细胞实验数据，计算拟合曲线拐点T和半最大值t1/2两个特征参数。发现[Ca2+]i、ΔΨm、溶酶体pH曲线的拐点（分别记为TCa、Tmito和Ti）数值上满足TCa
本文的研究将荧光显微成像技术应用于具体活细胞荧光观测和量化分析中，并将为活细胞生命科学提供有利的技术和方法。
Living cells are the basic structural units and functional units of organisms.Therefore, real-time dynamic observation in single living cells is of great importance inlife sciences and quantitative analysis the experiment data in depth is helpful to revealthe information of life sciences. In this thesis, a real-time multifunctional fluorescencemicroscopic imaging system as well as relevant image processing methods is improvedfor long term living cells observation. The studies of early apoptosis on thymocytes andthe spontaneous and synchronous intracellular Ca2+oscillations of cultured hippocampalneuronal networks are carried out in the system.
First, the real-time multifunctional fluorescence microscopic imaging systembecame more suitable for long term living cells observation after the installation ofincubator and the development of relevant image processing methods.
Secondly, a series of changes in thymocytes early apoptosis induced byS-nitrosoglutathione (GSNO) were studied. During the early apoptosis, the increase ofintracellular free calcium ion concentration ([Ca2+]i), the loss of mitochondrialmembrane potential (ΔΨm), the decrease of lysosomal pH, cytoplasmic acidification ofapoptosis cells were detected. The kinetic features were quantitatively analyzed andcompared by fitting the experiment data. The mathematical parameters, inflection pointand half-max effect point were proposed to analyze the fitting curve. The resultsrevealed that the inflection point of lysosomal pH always appeared prior to that of ΔΨmand posterior to that of [Ca2+]iin a certain GSNO concentration induced. Half-maxeffect point was also displayed the similar results. Such quantitative analyses ofreal-time observations are useful for interpreting the sequence of the biological eventsoperating in GSNO-induced thymocyte apoptosis and provide important clues forapoptotic signaling pathway studies.
Finally, the modulation of different compatibilities of Chinese herbal medicine onsynchronized Ca2+oscillations in cultured hippocampal neuronal networks were carriedout. We investigated and quantitative analyzed the modulation effects of lowconcentration and different compatibilities on synchronized Ca2+oscillations of piperineand deoxyschizandrin, deoxyschizandrin and schisandrin B in cultured hippocampalneuronal networks. The results suggest that different compatibilities have different inhibition on the synchronized Ca2+oscillations. The inhibitory effect on the frequencyis irrelevantly to that on the amplitude. A calcium oscillation model of nerve cells wassetup and the effects of its seven parameters were analyzed.
This thesis is a specific application of fluorescence microscopic imagingtechniques in living cells and quantitatively analyses and supposed to provideadvantageous techniques and methods to studies on living cells.
引文
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