【作者】 卢韵碧；

【导师】 魏尔清； Andreas Reichenbach； Josef A. K(?)s；

【作者基本信息】 浙江大学 ， 药理学， 2006， 博士

【摘要】 细胞的生物力学性质(biomechanical property)在机体的发育、正常功能发挥以及疾病过程中起重要的作用。对其他组织的力学性质的研究已有许多报道，如骨组织在力负荷作用下的生长、血细胞变形游走需要细胞具有特殊的力学性质(mechanical property)等；然而，仅在最近的几年才有少数的报道涉及探讨中枢神经系统的力学性质。实际上，力学现象在中枢神经系统中也起很重要的作用，如神经细胞在神经系统的发育过程中受牵拉变形(典型的例子是脊髓末段马尾的形成)。细胞在力的作用下如何变形，决定于细胞对力的感知(mechanosensing)和转导(force transduction)，以及细胞内在的力学性质。目前，我们关于神经细胞的力学性质的知识十分有限，由于这个原因，关于神经胶质细胞力学性质的两个完全相反的预言被广泛接受：(1)神经胶质细胞(glia，在希腊语中意为胶水)的特性是粘性的(viscous)；(2)神经胶质细胞在神经组织结构中起“支柱”(support pillar)作用，其特性是很硬的(stiff or hard)，如视网膜Müller胶质细胞。这些细胞的生物力学性质可能影响神经组织结构的形成以及神经元-神经胶质细胞间的交互作用(neuron-glia cross talk)。为此，在本研究中，我们将初步探讨中枢神经系统的神经元以及神经胶质细胞的力学性质。我们利用扫描力显微镜法(scanning force microscopy)定量检测了神经细胞的粘弹性(viscoelasticity)。利用扫描力显微镜法检测细胞粘弹性，最适宜的方法是检测过程中，在探头(cantilever)的直流的信号上叠加一个高频低振幅的振荡信号进行检测。我们在3个不同的频率下(30，100，200 Hz)检测了自成年哺乳动物中枢神经系统的两个区域急性分离而得的神经细胞：(1)从海马分离的锥体神经元(pyramidal neuron)以及星形胶质细胞；(2)从视网膜分离的中间神经元(interneurons，即双极细胞[bipolar cells]和无长突细胞[amacrine cells])，以及Müller胶质细胞。结果以构成复数型粘弹性常数K~*(complex viscoelastic constant)的两部分描述，即实数部分K′(此部分反映细胞的弹性／硬度[elasticity／stiffness]和储存能量反应[elastic storage response])和虚数部分K″(此部分反映细胞的粘性[viscosity]和耗散能量反应[viscous loss response])。此外，我们还首次报道了细胞各部分的泊松比(Poisson’s ratio)，该指标反映物体(细胞)在力的作用下横应变与纵应变之间的比值。我们发现：(1)神经元的弹性常数约为1000 Pa，低于已报道的其他细胞的弹性常数(如纤维母细胞)，这样，纤维母细胞的硬度是神经元的两倍；(2)神经胶质细胞比神经元软一倍；(3)神经元和神经胶质细胞的K′均高于他们的K″，即相比于粘性，他们的弹性占优势。这说明胶质细胞具有象屈从性的软弹簧(very soft and compliant spring)一样的特性，神经元和神经胶质细胞展示了象很软的橡胶一样的力学性质。我们实验室平行进行的组织和单细胞水平的生物物理实验结果，和以上结果一致，如组织切片流变学(bulk rheological measurement)和激光细胞牵拉器(optical cell stretcher)检测，以及透射电镜的结果。另外，我们以视网膜Müller胶质细胞为例，检测了单个神经细胞不同部位的粘弹性。据Müller细胞的形态，沿其长轴(从Müller细胞的终足到巩膜)分为：终足(endfoot)，内突起(inner process)，胞体(soma)，外突起(outer process)。Müller细胞具有复杂的细胞骨架且细胞各部分有不同的细胞器。除肌动蛋白纤维丝(actin filaments，其构成整个细胞的‘皮质’cortex)外，Müller细胞的终足含有致密的滑面内质网，内突起主要含中间丝，胞体主要含细胞核，外突起主要含微管。我们的结果显示，Müller细胞的突起比其终足和胞体软，胞体是该细胞最硬的部分。海马锥体神经元(其胞体主要含细胞核，其突起主要含微管)的检测结果显示，神经元的突起比胞体软。这表明单个神经细胞不同部位的粘弹性可由细胞的亚细胞结构决定，但这还需要进一步实验证实。总之，我们的结果显示，中枢神经系统的神经元和神经胶质细胞的力学特性是很软的(具橡胶样弹性的，‘rubber elastic’)且有相当大的粘性(耗散能量的，‘dissipative’)；神经元和神经胶质细胞的粘弹性以它们的弹性占优势；神经胶质细胞比神经元更软(即更容易变形)。因此，我们认为，神经胶质细胞的作用既不是中枢神经组织结构的“支柱”，也不是粘合神经元的“胶水”；实际上，神经胶质细胞象一种减震材料一样紧紧围在神经元周围，在受到机械性损伤时，神经胶质细胞可以防止或减轻神经元的受损。更重要的是，神经胶质细胞构成了一种很软的、适于神经元及其突起生长的底物，因此，神经胶质细胞的这种生物力学性质利于神经元的可塑性形成(neuronal plasticity)。更多还原

【Abstract】 The biomechanical properties of animal cells were shown to play important roles in the development and functioning of the body, and also in the pathological mechanisms of many diseases. However, only in recent years there are a few studies on mechanics in the central nervous system (CNS), whereas other tissues have been extensively studied. It became clear that such mechanical phenomena may be very important also for the CNS, where, for instance, neuronal cells are being stretched during ontogenetic development (the formation of the cauda equina may serve as an example to illustrate the problem). The mechanical properties of neural cells may contribute to neural tissue structure and even to neuron-glia cross talk. Thus, in the present study we investigated the mechanical properties of glial cells and neurons in the CNS.We measured the viscoelasticity of neural cells by using scanning force microscopy (SFM). Since the cells are viscoleastic, dynamic measurements wereperformed by introducing a high-frequency, low-amplitude oscillation to the cantilever. All experiments were performed on acutely dissociated cells from two different CNS regions: (i) pyramidal neurons and astrocytes (glial cells) of the hippocampus, and (ii) bipolar and amacrine cells (i.e., interneurons) and Müller (glial) cells of the retina. There are two components of the complex viscoelastic constant K~*, of which K’ reflects the elastic storage response of the cells and K" reflects the viscous loss response of the cells. These constants, as well as the Piosson’s ratio v, were obtained for single cells at three different frequencies (30, 100, and 200 Hz).By applying this method, we found that the elastic constant of neurons is only about 1000 Pa. This is less than that of other cells; for instance, fibroblasts are two times suffer than neurons. Furthermore, glial cells were found to be about two times softer than neurons; thus, they behave as a type of very soft and compliant spring. For both types of cells, K’ is higher than K"; this means that, compared to the viscous property, the elastic property is dominant. Thus, CNS-cells display the features of a very soft elastic rubber. Parallel biophysical and electron microscopal studies in our laboratory confirmed the data on the levels of tissue sections and of single cells.To figure out the viscoelastic properties of different cellular compartments, retinal Müller cells - as prototypical glial cells - were subjected to measurements along their length, comprising various parts from endfoot, inner process, soma, and outer process up to the sclerad end. Müller cells not only posses a complex cytoskeleton but contain different cellular organelles in these different cellular compartments. In addition to actin filaments which form a "cortex" of the entire cell, the endfeet of Müller cells are densely packed with smooth endoplasmic reticulum, the inner processes contain intermediate filaments, the somata are packed with the nucleus, and the outer processes contain microtubules. The results showed that both Müller cell processes were significantly softer than the soma and the endfoot. A similar situation was found in pyramidal cells, whose soma contains the nucleus and the processes are packed with microtubules. In these cells, the apical dendrite was significantly softer than the soma. However, it remains to be elucidated why the local mechanical properties of CNS cells vary depending on their subcellular structure(s).In conclusion, the results suggest that in general, both neurons and glial cells are very soft (’rubber elastic’) and considerably viscous (’dissipative’). Glial cells are even softer than neurons. These results allow the conclusion that glial cells do neither serve as support cells nor as glue. Rather, they may act as a shock absorbing material embedding the neurons and diminish the damage of the neurons in case of mechanical trauma. Furthermore, they constitute a very soft (and, thus, optimal) substrate for the growth of neuronal cells and their processes. Therefore, this mechanical feature of the glial cells may support neuronal plasticity.更多还原