利用一個隔室化的神經培養裝置產生不同挫傷程度的軸突以做藥物篩選

Using a compartmentalized culture device to generate axons at different levels of contusion injury for drug screening

何勃衛(Bo-Wei Ho)

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(2018年)

摘要

中樞神經系統的再生能力很弱，若是受到損傷，通常會是嚴重且不可逆的傷害。除了神經退化性疾病之外，受到外力衝擊而造成的神經損傷也是一大宗。在美國，一年之中，平均每一百萬人就有54人承受著脊椎損傷之苦。，在台灣平均每年每一百萬人中就有約151人承受著脊椎損傷之苦，更甚美國。脊髓損傷全球的發生率範圍在每百萬人13.0-163.4件之間，主要原因大多為車禍及摔倒。尋找神經軸突的再生與導引，以及相關調節軸突功能的藥物，對於醫治軸突損傷相關疾病的方法已是迫在眉睫。雖然目前對再生方面的了解有限，但隨著對神經軸突損傷的研究越多越廣，在深入理解傷後反應及再生機制後找到治療的機會是可以樂觀期待的。
本研究中使用本實驗室先前開發的神經細胞培養裝置，將直徑約為10 μm的細胞本體留在PCTE膜上，而軸突在PCTE膜下方第二層PDMS膜的管柱空間中生長，相似於腦軸突在脊椎中之排布，我在PCTE膜與PDMS膜間插入細針，藉此對軸突引發不同程度的拉伸損傷，並且實際檢驗是否能夠利用此裝置作為神經軸突保護藥物的篩選工具。我們希望藉此研究建立一套有助於神經軸突保護藥物的初期篩選系統，有效篩出富有療效潛力的藥物，然後進入動物實驗，期望減少實驗動物的使用數量，為全世界面臨脊椎損傷所苦的病患找出有效的治療方法，提升病患的生活品質、信心與尊嚴。

關鍵字

神經培養 ； 軸突 ； 挫傷 ； 藥物篩選 ； 隔室化

並列摘要

In the mammalian central nervous system, most injured axons do not regenerate. In addition to neuronal degenerative diseases, neuronal injuries are also often caused by mechanical forces. In the USA, approximately 54 cases per one million people suffer from spinal cord injuries each year. The incidence of traumatic spinal cord injury is about 151 cases per million people in Taiwan and is higher than that of the USA. In world-wide, the incidence of traumatic spinal cord injury is in a range between 13.0 and 163.4 cases per million people with the leading causes of motor vehicle crashes and falls. Finding cure for neuronal injuries is urgently needed. Although our understanding of the mechanisms of axonal regeneration is still incomplete, with rapid progresses in the field, finding a cure for spinal cord injury is a feasible task.
We utilize polycarbonate (PCTE) multi-pore cell filter and a hole-filled polydimethylsiloxane (PDMS) membrane to produce a compartmentalized neuronal culture devices. When grown in this device, the cell bodies are separated from the neurites by the PCTE membrane. The PDMS membrane containing holes of 500 μm in height, serve to separate axons from dendrites due to the latters’ shorter length. Neurons growing in such a device mimic those in the brain and spinal cord in their spatial arrangements. I could induce injury of different degrees to axons by inserting a fine needle between the PCTE and PDMS membrane. I also tried to test the feasibility of using this system for screening drugs which can protect axons from contusion injury. To the end, I hope to use this system as a spinal cord contusion injury model for drug screening and for uses in basic and clinical research. The use of this cell culture system will help reduce the experimental animals used in such studies.

並列關鍵字

neuronal culture device ； axon ； contusion injury ； drug screening ； compartmentalized

參考文獻

Allen AR (1911) SURGERY OF EXPERIMENTAL LESION OF SPINAL CORD EQUIVALENT TO CRUSH INJURY OF FRACTURE DISLOCATION OF SPINAL COLUMN : A PRELIMINARY REPORT. JAMA LVII(11):878-880.

Allen AR (1914) Remarks on the Histopathological Changes in the Spinal Cord due to Impcat. An Experimental Study.

Alto LT, Havton LA, Conner JM, Hollis ER, 2nd, Blesch A, Tuszynski MH (2009) Chemotropic guidance facilitates axonal regeneration and synapse formation after spinal cord injury. Nat Neurosci 12:1106-1113.

Anderson TE (1982) A controlled pneumatic technique for experimental spinal cord contusion. J Neurosci Methods 6:327-333.

Bar-Kochba E, Scimone MT, Estrada JB, Franck C (2016) Strain and rate-dependent neuronal injury in a 3D in vitro compression model of traumatic brain injury. Sci Rep 6:30550.