研究中樞神經細胞軸突之新型隔室化細胞培養裝置

A compartmentalized culture device for studying the axons of CNS neurons

鄭美雲(Mei-Yun Cheng)

指導教授 ： 張兗君

國立清華大學/生命科學院/分子醫學研究所/博士(2017年)

摘要

來自其他神經元細胞的訊號，經由樹突傳入後，透過神經元的細胞本體整合，再將整合後的訊號經由軸突傳遞，並通過突觸發送到適當的標的。許多急性與慢性神經性疾病會引起軸突損傷或變性，導致嚴重後遺症。因此，研究軸突導向與軸突再生，對於發展有效的臨床治療是非常重要的。在這篇研究中，我將報告一種新型隔室化細胞培養裝置，可用於研究中樞神經系統（CNS）神經元軸突的分子細胞生物學和軸突功能。
此隔室化細胞培養裝置可用於將中樞神經元的軸突與細胞本體、樹突分離。該裝置由隔膜將之分為頂部與底部兩個隔室所組成，隔膜藉由微通道填充的聚二甲基矽氧烷（PDMS）膜的頂部上連接多孔聚碳酸酯道蝕（PCTE）過濾膜而構建。隔膜的表面和微通道，分別塗覆和填充支持神經元生長與神經突移動的物質。當大鼠海馬神經元在頂部隔室中培養時，軸突和樹突都可以通過PCTE過濾膜中的孔並移行到PDMS膜的微通道中。在體外培養第8至9天後，唯獨軸突能生長抵達至下方隔室，軸突可以透過固定後的免疫螢光染色或實時監測來研究。每一個隔室化細胞培養裝置可以分離出大約3微克軸突蛋白質以供生化分析。此外，隔膜還可阻礙小分子在頂部和底部隔室之間的移動，因此上、下隔間可以分別接受獨立處理，觀察不同操作對於神經細胞本體與樹突或軸突的影響。接著，將討論如何應用此裝置，用在神經元軸突的各種研究和篩選不同試劑對於調節軸突功能的影響。結論，隔室化細胞培養裝置可以做為有效分離中樞神經軸突與中樞神經元的工具之一，並可用於對軸突進行深入的分子生物學分析，以及調節軸突功能的藥物快篩。

關鍵字

隔室化細胞培養裝置 ； 海馬迴神經元 ； 軸突 ； 軸突蛋白質

並列摘要

The cell body of neuron integrates various signals from dendrites, and the integral signals are transmitted by axons and sent to appropriate targets via synapses. Devastating sequels resulting from axonal injury and degeneration are common in many acute and chronic neurological diseases. The research in axonal guidance and regeneration is important to develop effective clinical treatments. Here, I report a novel compartmentalized culture device for studying the molecular and cell biology and functions of the axons of central nervous system (CNS) neurons.
The compartmentalized culture device allows the spatial separation of the somatodendrites and axons of CNS neurons. The device consists of two compartments separated by a septum constructed by attaching a porous polycarbonate track etch (PCTE) filter on top of a microchannel-filled polydimethylsiloxane (PDMS) membrane. The surface and microchannels of the septum are coated and filled, respectively, with materials that support neuron growth and neurite migration. When rat hippocampal neurons are cultured in the top compartment, both the axons and dendrites can migrate through the pores in PCTE filter and into the microchannels of the PDMS membrane. After 8-9 days in vitro, axons are the only processes that emerge from the septum and grow in the bottom compartment. Axons could be either studied by fluorescence immunocytochemistry after fixation or monitored in real-time. Axons containing ~3 µg protein can be isolated from each device for biochemical analyses. In addition, the septum also impedes the movement of small molecules between the top and bottom compartments. This feature allows the somatodendrites and axons of neurons, which occupy the top and bottom compartments of the device, respectively, to be manipulated independently. The potential applications of the device as a tool in diverse studies concerning neuronal axons and in screening reagents that regulate axonal functions have also been discussed. In conclusion, the compartmentalized culture device will be a valuable tool in separating axons from the central neurons for conducting in-depth molecular biology analyses of the axon and high-throughput screening for drugs regulating axonal functions.

並列關鍵字

Compartmentalized culture device ； Hippocampal neurons ； Axons ； Axonal proteins

參考文獻

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