利用細胞培養裝置分離並收集中樞神經細胞軸突以進行蛋白質體研究

Separation and enrichment of CNS axons for proteomic analyses by using a compartmentalized device

梁允聰(Leong, Wan Chong)

指導教授 ： 張兗君

國立清華大學/生命科學院/系統神經科學研究所/碩士(2016年)

摘要

在神經科學領域中研究的對象通常是為神經元細胞(Neuron)，神經元細胞主要的由細胞本體(Cell body或Soma)、樹突(Dendrite)及軸突(Axon)這三種主要部份所構成，其中軸突的作用主要為傳遞神經訊號至其他細胞，而在軸突轉譯及傳送所需要的蛋白質是近代重點研究的對象。研究神經細胞的軸突並不容易，這需要將軸突和細胞本體分離，因此已有開發多種神經軸突分離裝置來進行純軸突的研究。本文也延續使用本實驗室最新開發的神經軸突分離裝置來分離軸突，這種裝置有別於以往的裝置，能收集相較到更多的軸突，製作過程也相對容易。本文選用此裝置培養神經細胞並收集了單純軸突的蛋白質進行分析及定量。這裝置是一種雙層膜結構的垂直型分離裝置，將神經細胞培養在PCTE膜中，神經軸突會穿通PCTE膜並在PDMS膜上持續生長，在DIV17之後，拆除裝置，就可以對這兩層的膜進行免疫螢光染色以觀察軸突的生長狀況，在蛋白質分析實驗中，同樣在DIV17後，從PDMS膜上刮下軸突細胞，收集軸突的蛋白質；為分析單純軸突之蛋白質，本文使用了同為軸突分離裝置的神經基板裝置來培養神經細胞並分離出單純軸突作為參照，以其蛋白質濃度推算出本文裝置所收集的軸突蛋白質濃度，決定軸突的濃度後進行西方墨點法分析收集之軸突，以證實PDMS膜上所收集的軸突並無樹突的污染。結果表明，新開發的軸突裝置能分離的軸突數量上相較於以往的裝置有成倍的增長，並在蛋白質的層面上證明所收集的軸突也擁有高分離純度，也同時證實在未來也可使用此裝置分離軸突及細胞以進行更多軸突蛋白質的研究與分析。

關鍵字

神經 ； 軸突 ； 軸突分離裝置 ； 蛋白質體

並列摘要

Neurons are the primary structural unit of nervous systems. The basic structure of a typical neuron consists of a cell body, dendrites and an axon. Axons play a main role in the signal transduction to other connected neurons. Recently, researches of local translation in axons has become center stage. These researches need to use techniques allowing to physically separate the cell bodies and axons. Several such devices have been developed. In my thesis, I use a new device developed in our Lab which can be used to collect more axons and be fabricated easily. Rat hippocampus neurons were prepared to culture in this new device. This new device has a double layered structure. The first layer is a piece of PCTE membrane, and the second layer is a thin PDMS (Poly dimethylsiloxane) sheet perforated by many round holes. I grow hippocampal neurons on the PCTE membrane. Most of the axons grow through the PCTE membrane into the second layer, PDMS membrane. On 17 day-in-vitro, many axons migrate the length of holes and grow on the opposite surface. Axons on PDMS surface were detected by using fluorescence immunostaining. I collect axons from the PDMS surface by using a rubber scraper. The content of axons in the collected sample is determined by comparing to the axon samples produced by using another lab-on-a-chip device also developed in the laboratory. Western blotting is also used to verify whether the axon sample has free contaminations from soma to dendrites. The results indicated that the new developed device in the laboratory can be used to harvest pure axons from cultured rat hippocampal neurons with a yield of axons around 4 fold higher than the earlier lab-on-a-chip device.

並列關鍵字

neuron ； axon

參考文獻

黃琮凱. (2013). 發展一個分離中樞神經細胞軸突的細胞培養裝置. 國立清華大學分子醫學所.

CAMPENOT, R. B. (1977). Local control of neurite development by nerve growth factor. Cell Biology, 74, 4516-4519.

Ivins, K. J., Bui, E. T. N., & Cotman, C. W. (1998). b-Amyloid Induces Local Neurite Degeneration in Cultured Hippocampal Neurons: Evidence for Neuritic Apoptosis. Neurobiology of Disease, 5, 365-378.

Jang, J. M., Tran, S. H., Na, S. C., & Jeon, N. L. (2015). Engineering controllable architecture in matrigel for 3D cell alignment. ACS Appl Mater Interfaces, 7(4), 2183-2188. doi: 10.1021/am508292t

Kaplan, M. S. (2001). Environment complexity stimulates visual cortex neurogenesis death of a dogma and a research career. TRENDS in Neurosciences, 24, 617-620.