Institute of Molecular Medicine, National Tsing Hua University, Hsinchu, Taiwan, Republic of China.
Lab Chip. 2010 Mar 7;10(5):647-53. doi: 10.1039/b918217a. Epub 2009 Dec 23.
Axons are long, slender processes extending from the cell bodies of neurons and play diverse and crucial roles in the development and function of nervous systems. Here, we describe the development of a chip device that can be used to produce large quantities of axons for proteomic and RNA analyses. On the chip surface, bundles of axons of rat hippocampal neurons in culture are guided to grow in areas distinct and distant from where their cell bodies reside. Fluorescence immunocytochemical studies have confirmed that the areas where these axons are guided to grow are occupied exclusively by axons and not by neuronal somatodendrites or astroglial cells. These axon-occupied parts are easily separated from the remainder of the chip and collected by breaking the chip along the well-positioned linear grooves made on the backside. One- and two-dimensional gel electrophoresis and Western blotting analyses reveal that the axons and whole cells differ in their protein compositions. RT-PCR analyses also indicate that the axons contain only a subset of neuronal RNAs. Furthermore, the chip device could be easily modified to address other issues concerning neuronal axons, such as the molecular composition of the axon substructure, the growth cone and shaft, the degeneration and regeneration processes associated with injured axons and the effects of extrinsic molecules, such as axon guidance cues and cell adhesion molecules, on the axon. With these diverse applications, the chip device described here will serve as a powerful platform for studying the functional proteome of neuronal axons.
轴突是从神经元细胞体延伸出来的长而细的突起，在神经系统的发育和功能中发挥着多样化且至关重要的作用。在这里，我们描述了一种芯片设备的开发，该设备可用于大量产生轴突，用于蛋白质组学和 RNA 分析。在芯片表面，培养的大鼠海马神经元的轴突束被引导在与细胞体所在位置不同且远离的区域生长。荧光免疫细胞化学研究证实，这些被引导生长的轴突区域仅被轴突占据，而不是神经元胞体、树突或星形胶质细胞。这些被轴突占据的部分很容易与芯片的其余部分分离，并通过沿着背面制作的定位良好的线性凹槽将芯片折断来收集。一维和二维凝胶电泳和 Western blot 分析显示，轴突和整个细胞在蛋白质组成上存在差异。RT-PCR 分析还表明，轴突仅包含神经元 RNA 的一个子集。此外，该芯片设备可以很容易地进行修改，以解决其他与神经元轴突有关的问题，例如轴突亚结构、生长锥和轴突的分子组成、与损伤轴突相关的退化和再生过程以及外源性分子（如轴突导向线索和细胞粘附分子）对轴突的影响。有了这些多样化的应用，这里描述的芯片设备将成为研究神经元轴突功能蛋白质组学的强大平台。
