标题: 检验NuMA和TPX2蛋白在神经突型态发生中的功能
Examining the function of NuMA and TPX2 in neurite morphogenesis
作者: 陈雯馨
Chen, Wen-Shin
黄兆祺
Hwang, Eric
生物资讯及系统生物研究所
关键字: 分离海马回神经;微管相关蛋白;神经突;神经突发生;NuMA蛋白;TPX2蛋白;Dissociated hippocampal neurons;Microtubule-associated protein;Neurite;Neuritogenesis;NuMA;TPX2
公开日期: 2010
摘要: 神经系统的功能来自于神经元之间复杂的网络连结。为了形成正确的神经回路,每颗神经元之间的连结必须被适当地调控,以建立有效的神经网路。神经突发生 (neuritogenesis)能确保神经正确地连结,在神经突的发展过程中占有相当重要的地位。目前已知细胞骨架中的微管和微管相关蛋白 (microtubule-associated proteins)在神经突发生的调控中扮演了非常重要的角色。
NuMA和TPX2是两个在细胞分裂中很关键的微管相关蛋白,纺锤丝的集合和维持都需要NuMA,而产生稳定的双极纺锤丝 (bipolar spindle)则需要TPX2的参与。特别的是NuMA和TPX2不论在细胞间期或是有丝分裂后的细胞(post-mitotic cell, 例如神经元)里都存在于细胞核中。但NuMA和TPX2在神经系统中的功能目前仍不清楚。
先前的实验显示以P19所分化的神经细胞为实验材料,抑制神经细胞中的NuMA和TPX2会导致神经细胞长出较短的神经突。为了更进一步检验这个现象,后续实验以小鼠海马回神经细胞为实验对象,将能够转录出抑制NuMA或TPX2的shRNA质体转染进海马回神经细胞的初代培养 (primary culture)当中,并分析抑制NuMA和TPX2是否会改变神经突的生长。
我们发展出改良过的初代神经细胞培养方法,可培养出较健康的神经细胞,经过转染后其存活率亦较高。本次研究中发现NuMA和TPX2确实与神经突生成相关。在小鼠海马回神经元中抑制NuMA或TPX2皆会降低其神经突的数量但却不影响神经突长度。除此之外,在被抑制的神经细胞中表现外来的人类NuMA或TPX2可让神经突数目略微回升。这些结果显示NuMA和TPX2确实在神经突的形成(initiation)过程中扮演重要的角色。
A functional nervous system depends on the intricate connections between neurons. For neuronal circuits to be correctly wired, connections of each neuron must be properly regulated to establish an effective neural network. Neuritogenesis is an important neurite developmental process that ensures proper neuronal connections are established. It has been shown that microtubule cytoskeleton and microtubule-associated proteins are important regulators of neuritogenesis.
The microtubule cytoskeleton plays an indispensable role in each of the steps during neuritogenesis. Two microtubule-associated proteins, NuMA and TPX2 play critical roles in mitosis. NuMA is an essential mitotic component to establish and maintain focused spindle pole and TPX2 is required to generate a stable bipolar spindle. Interestingly, NuMA and TPX2 localize to the nucleus during interphase or post-mitotic cells (such as neurons). However, the non-mitotic functions of NuMA and TPX2 remained elusive.
A previous study showed that suppressing NuMA or TPX2 in P19-differentiated neurons resulted in shortens neurites. To confirm this effect, mouse primary hippocampal neurons are transfected with plasmids which express EGFP as a report gene and also can be transcribed into shRNA targets NuMA or TPX2. Then neurons are analyzed if NuMA or TPX2 suppressed neurite elongation.
In this study, we established a novel method to culture dissociated mouse hippocampal neurons. Our method significantly simplifies the preparation while produces healthy and long-lived neuronal cultures. We also confirmed that NuMA and TPX2 indeed involve in neurite outgrowth. Suppressing NuMA or TPX2 in mouse hippocampal neurons resulted in reduced neurite number. Moreover, overexpressing human NuMA and TPX2 partially rescued the reduction of neurites. Those results suggest that both NuMA and TPX2 play important roles in neuritogenesis.
URI: http://140.113.39.130/cdrfb3/record/nctu/#GT079851502
http://hdl.handle.net/11536/48198
显示于类别:Thesis