發展氮化鎵晶片對於培養神經細胞、神經幹細胞以及嗜鉻細胞之研究

Abstract

神經細胞在體內生長須靠neurotrophic factors 及神經傳入的刺激才能良好生存。小腦顆粒神經細胞為高度均一的神經培養模式，一直被廣泛應用於神經細胞生長發育及凋亡機制的研究。而神經幹細胞則是具有多向分化潛能的功能性細胞，具有修補中樞神經的潛力。體外培養所使用的細胞培養基材，一直是影響細胞培養的一大因素，因此本研究中探討生物活性分子(聚離氨酸)、 矽晶片、氮化鎵、砷化鎵及一般常使用的細胞培養盤在培養神經細胞或神經幹細胞時所造成細胞貼附及生長的差異性。另外為了了解造成這些差異性的原因，本研究選用嗜鉻性神經細胞瘤細胞株，嘗試從訊息傳導路徑有關的蛋白質做相關的分析，藉由蛋白質表現量的不同來解釋細胞行為的差異。 第一章介紹中樞神經細胞、神經幹細胞、嗜鉻性神經細胞以及所使用之半導體材料對於培養細胞之應用與比較。 第二章在探討神經細胞分別在氮化鎵、矽基材以及一般培養皿培養三到六天後細胞之貼附與分化的程度，根據光學照片結果顯示，在培養三天後之神經細胞大多能在各個基材上形成細胞貼附與分化。而在培養到六天時，在氮化鎵基材的作用下，神經細胞會呈現更高度的分化，矽基材上在貼附及分化上皆呈現下滑的趨勢，另外在乳酸脫氫脢量測細胞貼附數量的檢測，也呈現相同的趨勢。 第三章則是探討氮化鎵培養神經細胞在長時間下的神經突觸功能表現，結果顯示氮化鎵相對於聚離氨酸與一般培養皿具有較佳保持神經突觸功能的情形，而在相對細胞死亡測試實驗中，也顯示氮化鎵較能促進細胞的存活，此結果可能由於氮化鎵基材在早期磷酸化了蛋白質Akt。 第四章主要探討神經幹細胞在氮化鎵和聚離氨酸上的分化情形，培養結果顯示在這兩種基材上皆能讓細胞貼附及分化，相對於聚離氨酸，氮化鎵更能讓神經幹細胞往神經細胞的路徑去分化，此結果可能導因於glycogen synthase kinase-3β (GSK-3β)活性的抑制，而進入神經細胞的分化。另一方面，氮化鎵具有讓神經幹細胞長時間貼附與促進細胞存活的能力。 第五章則是以嗜鉻細胞做為研究神經細胞訊息傳導路徑模型，實驗結果顯示氮化鎵可能會經由Akt/GSK-3β/ caspase-3路徑，阻止神經細胞的凋亡，進而促進神經細胞的存活，在乳酸脫氫酶測試上也呈現相同的趨勢。 第六章總結本研究的貢獻，本研究重要的發現在於三五族半導體氮化鎵單晶所造成的Lewis acid-base性質能夠促進神經細胞以及神經幹細胞的貼附及分化，使得它在神經生物晶片與細胞體外培養系統發展上更具競爭力。

Neurons can only grow well in body by means of neurotrophic factors and afferent neuron stimulus. Cerebellar granule neurons belong to highly uniform neural culture model, and have been widely used in neuron growth and apoptosis mechanism researches. Neural stem cells are functional cells with multi-differentiation potency, having potential of repairing central nerve. Cell culture substrate for in vitro cultivation has always been a major factor in cell culture. This study examined cell adhesion and differentiation difference when adopting bioactive molecule-polylysine, silicon chip, GaN, GaAs and commonly used cell culture dish (polystyrene) as substrate of cultivating neurons or neural stem cells respectively. In addition, to understand the cause of such differences, this study selected neuronal pheochromocytoma strain was selected in to analyze signal transduction pathway, and cell behavior variation can be attributed to protein expression difference. Chapter 1 introduces application and comparison of central neuron, neural stem cell, chromaffin neuron and semiconductor material used on cell culture. Chapter 2 discusses neuron adhesion and differentiation after cultured on GaN, silicon substrate and tissue culture polystyrene (TCPS) for 3-6 days. As shown by optical micrographs, after cultured for three days, most neurons could form cell adhesion and differentiation on every above substrate. And after cultured for six days, neurons on GaN substrate showed more differentiation, neurons on silicon substrate reported deteriorated adhesion and differentiation. Furthermore, cell adhesion degree inspection on lactate dehydrogenase showed the same trend. Chapter 3 describes the long-term synapsis performance of GaN cultured neuron, showing that, GaN has better retention of synapsis function, as compared to polylysine and TCPS. Relative cell death test also showed that GaN could better promote cell survival, probably due to phosphorylation of Akt on GaN substrate in earlier time. Chapter 4 examines neural stem cell differentiation on GaN and polylysine respectively. Cultivation showed that both substrates could enable cell adhesion and differentiation, GaN was more likely than polylysine to enable neural stem cell differentiation along neuron path, probably due to inhibited activity of glycogen synthase kinase-3β (GSK-3β). On the other hand, GaN is capable of promoting long term adhesion of neural stem cell and cell survival. Chapter 5 takes chromaffin cell as the model of research on neuron signal transduction pathway. The experiment found that, GaN may suppress neuron apoptosis via Akt/GSK-3β/caspase-3 path, and thus promote neuron survival; lactate dehydrogenase test indicated the same trend. Chapter 6 concludes the contribution of this study that Lewis acid-base property derived from Group III/V semiconductor GaN single crystal could promote adhesion and differentiation of neurons and neural stem cells.