【作者】 周炜；

【导师】 骆清铭；

【作者基本信息】 华中科技大学 ， 生物医学工程， 2007， 博士

【摘要】 中枢神经系统中主要存在两类细胞:神经元和神经胶质细胞。通过记录电信号,人们能够准确地记录神经元的活动。但是神经胶质细胞作为一类非电兴奋型的细胞,仅靠电信号很难反映出它们的功能——这也是长期以来胶质细胞不被大家关注的原因之一。但是,从数量上来看,神经胶质细胞却是神经元的数倍,这提示神经胶质细胞可能在神经活动中发挥着更大的作用。近十年来,随着光学成像和光学标记方法的发展,胶质细胞在神经活动中的重要作用正在逐步被揭示。显微镜的发明将生命科学的发展引入了细胞水平。高分辨率、高对比度和高成像深度成为目前显微成像技术发展的目标。本文使用双光子激发扫描显微术和全内反射荧光显微术来研究了神经胶质细胞的形态与功能。在大鼠急性分离的海马脑片上,根据电生理和免疫组化的结果,区分了四种胶质细胞:星型胶质细胞,少突胶质细胞、NG2胶质细胞和小胶质细胞。通过电极注射荧光染料和双光子成像,观察了四种胶质细胞在正常生理状态下的形态;并进一步探索了它们的功能。对于星型胶质细胞,使用双光子解笼锁钙离子,发现在急性海马脑片上,星型胶质细胞的胞内钙升高能导致血管的舒张。对于少突胶质细胞,通过三维扫描成像,揭示了海马CA1区少突胶质细胞特有的突起分布形式:分为两种走向,呈十字分布。对于脑片中的NG2细胞,观察到其特有分裂方式:胞体一分为二,而突起按照在胞体上的位置分属不同的子细胞。利用飞秒脉冲激光刺激培养的星形胶质细胞,诱导了细胞内的钙升高及随后产生的钙波。不同的光强能诱导星型胶质细胞的产生两种钙振荡模式:短时的钙升高(即calcium spike,定义为S型)和高且可持续的钙升高(定义为H型)。光刺激后胞外的PI染料的进入,高数值孔径物镜观察到细胞膜的可逆变化及胞外钙离子的依赖性证实了光致穿孔效应是导致钙升高的原因。光穿孔诱导的钙升高能够在星型胶质间传播;药理学证据证实这种钙波是通过细胞释放的ATP介导,激活P2Y受体来实现的。同时在混合培养的细胞上,H型钙升高会引起附近神经元的同步钙振荡而S型则不会引起任何响应。以上结果说明,基于飞秒脉冲激光的光穿孔能在星型胶质细胞上诱导钙升高,从而导致P2Y受体依赖的钙波。而胶质细胞的钙波能诱导神经元产生同步的钙振荡。使用全内反射显微术研究了培养星型胶质细胞溶酶体的胞吐作用。使用TIRFM观察FM染料标记的溶酶体,发现星型胶质细胞的溶酶体有三种不同的释放现象,即膜上溶酶体的完全释放(full exocytosis from old puncta),新上膜溶酶体的完全释放(full exocytosis from new puncta)和部分释放模式(”kiss and run”)。在ATP刺激时,星型胶质细胞溶酶体的释放仅表现部分释放模式;而在缺氧条件下诱导了大量的溶酶体上膜,同时几乎所有在膜上的溶酶体都会被释放。更多还原

【Abstract】 The central nervous system is consisted of neuron and glial cell. By recording electronic signals, the neural activities could be described precisely. As a non-excitatory cell in neural system, however, glial cell is hardly touched their functions by electronic signal; that is why glial cells have not received neuroscientists’attentions. Moreover, several to ten times to neuron in number in central nervous system, glial cell have implied to exert more functions in neural system. At a last decade, by advances in optical imaging and labeling techniques, the functions of glial cell will have been understood.The invention of microscope brings the life science research to cellular level. High resolution, contrast and penetration are the hot topics in development of optical imaging techniques. Here, by using two-photon laser excitation scanning microscopy and total- internal reflection microscopy, the morphology and function of glial cell were investigated.In CA1 region of hippocampal slices, four types of glial cell were identified by electrophysiological characteristics and immunocytochemical staining: astrocytes, oligodendrocyte, NG2 (a chondroitin sulfate proteoglycan) positive cells and microglia. By pipette-injected dyes and two-photon laser scanning microscopy (2PLSM), their shapes were observed. Further, their functions were explored : For astrocyte, by two-photon induced uncaging calcium, the astrocytic calcium elevation induced the vasodilatation. For oligodendrocyte in CA1 region of hippocampal slices, by 3-dimensional imaging, their processes showed distinct distribution: about two third of all the processes (67.5±12.0%) are parallel to Sch fiber and the others were vertical to them. For NG2 glial cell, the distinct mitosis mode was observed: the cell body divides into two, the processes belong to the daughter cells according to their localization in cell body.Stimulated astrocyte by femtosecond pulse laser induced the intracellular calcium elevation and followed intercellular calcium wave. Under the different powers, astrocytes displayed two modes of calcium elevation: calcium spike (named S-type in following) and higher and sustained calcium elevation (named H-type in following). Some evidences, from PI penetration, membrane reversible shift under the high numerical aperture objective and extracellular calcium-dependent results, demonstrated that photoporation effects were causes to intracellular calcium elevation. Photoporation induced calcium elevation could spread among astrocytes; from pharmacological results, this calcium wave is mediated by ATP from astrocyte released and activated the P2Y receptor in adjacent astrocytes. Moreover, in mix culture of neuron and astrocyte, the H-type calcium elevation in astrocyte could induce neuronal synchronized calcium oscillation, whereas no any responses to S-type。In conclusion, the photoporation based on femtosecond pulsed laser could generate calcium elevation in astrocyte, and induce P2Y receptor-dependent calcium wave. The calcium waves in astrocytes lead the neuronal synchronized calcium oscillation.Investigate lysosome exocytosis revealed by TIRFM. By FM dye labeled lysosome and TIRF imaging, three types of release modes of lysosomes in astrocyte were showed: full exocytosis from old puncta mode, full exocytosis from new puncta mode and”kiss and run”mode. Physiological stimulation by ATP could induce partial exocytosis, whereas pathological stimulation by an ischemic insult (KCN) induced full exocytosis of lysosome.更多还原