臺灣博碩士論文加值系統

微環境的改變會影響幹細胞的生長調控，幹細胞在適當的微環境作用下，藉由貼附因子及分子訊息的傳遞，可維持幹細胞的自我更新與分化能力。本研究探討以微環境調控神經幹細胞的分化行為，進而應用在建立神經網絡與實時量測的晶片系統。神經幹細胞分化的促進、神經元的誘導及功能性突觸的形成是目前神經組織工程發展的重要需求。我們利用神經幹細胞具有自我更新、增生與分化為具功能性細胞的能力，藉由聚電解質多層膜基材表面改質、圖形化生長導引，以及氧化銦錫(Indium Tin Oxide, ITO)電極的偵測與刺激，建立即時的神經網絡刺激與分析平台。
聚電解質多層膜具有可調控帶電特性、厚度與表面剛性等性質。過去我們已發現，適度的材料堆疊可誘導神經幹細胞分化為具功能性的神經元。本研究在具有導電特性的氧化銦錫塗層玻璃上，逐層堆疊帶正電的聚離胺酸(Poly-L-Lysine, PLL)與帶負電的聚麩胺酸(Poly-L-Glutamic Acid, PLGA)，並探討此培養微環境對於神經幹細胞增生與分化之影響，並將其與傳統電場的電刺激方式進行比較。結果發現，聚電解質多層膜與電刺激皆可以促進神經幹細胞的分化，在聚電解質多層膜條件為(PLL/PLGA)7.5與(PLL/PLGA)8的條件下，會有較長的神經軸索與較高比例的具活性神經元存在；以FM1-43測試突觸活性，在聚電解質多層膜的條件中，亦可發現有較佳的突觸活性。
接著，為了進行神經幹細胞圖形化生長導引，我們使用SU-8負光阻，以光蝕刻製程製作微型流道，使神經幹細胞的神經軸索生長方向能夠受到導引，以形成神經網絡。藉由微型流道的控制，以及聚電解質多層膜的刺激，使得神經幹細胞得以在此培養平台上能夠促進生長並調控其生長方向。為了進一步建構即時偵測與分析平台，將導電性ITO塗層以光蝕刻製程製作微電極，並利用電阻抗量測技術，以即時量測任兩顆神經細胞球之間神經軸索的連接。此外，藉由ITO電極達到可對單一神經細胞球進行電刺激的功能，藉此觀察聚電解質多層膜與電刺激的加成效果。並藉由神經網絡的連接，觀察單一神經細胞球的電刺激，可觀察不同階層細胞球的訊息傳遞。結果顯示此晶片可利用電阻抗值量測進行即時偵測、分析神經元的分佈狀況，以利於監控與紀錄，透過分析電阻抗值和參考阻抗值，可以即時判定在兩個神經細胞球之間的神經軸索是否已經連接。
本研究利用微環境的改變，調控神經幹細胞的分化行為與功能性突觸生長。在神經幹細胞誘導部分，以ITO導電性基材、聚電解質多層膜及SU-8圖形化導引達到刺激神經幹細胞分化生長以及調控其分化頃向，並成功促進功能性神經元分化。進一步以ITO玻璃製作微型電極，利用ITO的導電性，可對細胞進行可控制範圍的電刺激，並以阻抗分析儀分析細胞分佈狀況，成功建立即時的細胞刺激與偵測平台。此晶片未來可應用於神經生長的即時檢測和分析，並輔助建立神經退化性疾病的體外模式與藥物測試。

Stem cell growth are regulated by microenvironment effect. Under appropriate microenvironment, maintenance of stem cell self-renewal and differentiation ability could be achieved by regulating of adhesion molecular and specific signal pathways. In this study, cell differentiation behavior of neural stem/progenitor cells (NSPCs) was controlled by regulating the microenvironment, and NSPCs further establish a neural network and real-time measurement of the chip system. Promotion of NSPCs differentiation, induction of synapses and the formation of functional synapses are important requirements for nerve tissue engineering currently. NSPCs possessed the ability of self-renewal, proliferation and differentiation. By polyelectrolyte multilayer (PEM) films, substrate surface modification, graphic growth guidance, detection and stimulation by indium tin oxide (ITO) electrodes, a real-time analysis and stimulation platform for neural network were established.
PEM films has many properties, such as regulatory charging characteristics, thickness and surface stiffness. Our previous study has revealed that NSPCs were induced into functional neurons by layer by layer assembling of PEM films. Herein, a conductive material, ITO coated glasses, was used as a base substrate and were stacked layer-by-layer by using Poly-L-Lysine (PLL) and Poly-L-Glutamic Acid (PLGA). The effects of ITO-PEM films on brain NSPCs proliferation and differentiation were investigated, and the effects were compared with the traditional electrical stimulation culture system. It was found that PEM films and electrical stimulation are able to promote the differentiation of NSPCs. But under the condition of PEM films (PLL/PLGA)7.5 and (PLL/PLGA)8, cells demonstrated longer synapse and higher percentage of active neurons. In addition, the activities of functional synapse were detected by FM1-43 and it revealed that synapse function of cells culture on PEM-ITO surface are higher than ITO surface.
In order to carry out the guidance of NSPCs, SU-8 negative photoresist were used by photo-etching process to produce microchannels. By using SU-8 microchannel, the direction of neurites outgrowth of NSPCs is capable of being guided and the neural network can be formed. By controlling the microchannels’ pattern as well as stimulating by the PEM films, the NSPCs could be cultured on this platform, differentiation were promoted and the growth direction were regulated. In order to construct a real-time detected and analyzed platform, ITO glasses were used to made microelectrodes by photo-etching process to measure the connection of neurites between two neurospheroid by using electrical impedance analysis. By ITO electrodes, each single neurosphere can be stimulated electrically. On this platform, the synergistic effect of PEM films and electrical stimulation could be observed. By neural network connection, the effect of neighboring neurospheroid could be observed by a single neurosphere electrical stimulation. By impedance analyzer, ITO electrode could be used for cell instant detection and analysis of synapses distribution, and to facilitate the real-time monitoring and record. By analyzing impedance values and the reference impedance value, it's possible to real-time determine whether the neurites have been connected successfully between two neurospheroid.
In this study, the differentiation of neural stem cells and growth of functional synapses could be controlled by regulating microenvironment. ITO conductive substrate, PEM films and SU-8 guide pattern could be used to stimulate growth and guidance differentiation tendency of NSPCs, this system may promote the collection of a large number of neurons. Furthermore, by using ITO conductive ability, the electrical stimulation on specific area of NSPCs spheroids could be achieved. By impedance analysis, the distribution of cells could be analyzed, and a real-time cell stimulation and detection platform are established. In the future, this platform can be applied to the real-time detection and analysis of neuritis outgrowth, and the in vitro model and drug testing model of neurodegenerative diseases can be established.

目錄
指導教授推薦書
口試委員會審定書
致謝 iii
摘要 iv
Abstract vii
目錄 x
圖目錄 xvii
表目錄 xxiv
縮寫一覽表 xxv
第一章 序論 1
1.1 序言 1
1.2 研究動機與目的 2
1.3 實驗架構 3
第二章 研究背景、原理與文獻回顧 4
2.1 微環境的影響因素 4
2.2 幹細胞及其特性 5
2.3 神經細胞與神經幹細胞及其特性 6
2.4 神經系統的生理 8
2.5 常見的神經疾病與損傷類型 9
2.5.1 常見的神經系統疾病 9
2.5.2 CNS與PNS 損傷類型 9
2.6 神經修復與再生 10
2.6.1 臨床醫學神經損傷治療 10
2.6.2 神經幹細胞療法 11
2.7 微環境對神經幹細胞的影響 12
2.8 聚電解質多層膜與應用 13
2.8.1 聚電解質多層膜特性及其應用 13
2.8.2 逐層鍵結對材料表面性質的影響 15
2.8.3 聚電解質多層膜應用於細胞培養及其影響因素 16
2.9 ITO塗層介紹與應用 17
2.10 氧氣電漿清洗與表面改質 18
2.11 神經網絡形成 18
2.12 細胞生長導引與微型通道 19
2.13 光阻材料與應用 20
2.14 阻抗分析法 21
2.15 阻抗分析法應用於細胞培養 22
2.16 ITO微電極 25
第三章 於ITO塗層玻璃基材上調控神經幹細胞生長 26
3.1 研究目標 26
3.2 實驗材料、方法與儀器 26
3.2.1 材料與藥品 26
3.2.2 儀器設備 30
3.2.3 氧氣電漿清洗與表面改質 32
3.2.4 聚電解質探討 32
3.2.4.1 正電聚電解質 32
3.2.4.2 負電聚電解質 32
3.2.4.3 PLL、PLGA溶液製備 33
3.2.4.4 聚電解質多層膜組裝 33
3.2.5 神經幹細胞萃取與培養 34
3.2.5.1 前置準備 34
3.2.5.2 分離細胞的過程 34
3.2.5.3 細胞繼代培養步驟 36
3.2.6 神經軸索長度量測與統計 37
3.2.7 免疫螢光染色與封片 37
3.2.8 細胞毒性分析 40
3.2.9 分化細胞型態鑑定與分化比例統計 40
3.2.10 突觸活性測試 41
3.2.11 電刺激系統結構 42
3.2.12 統計分析 43
3.3 結果與討論 44
3.3.1 神經幹細胞細胞分化形態 44
3.3.2 電刺激促神經幹細胞分化分析 44
3.3.3 聚電解質多層膜促神經幹細胞分化分析 46
3.3.4 電刺激與聚電解質多層膜促神經幹細胞分化比較 47
3.3.5 細胞毒性分析 47
3.3.6 免疫螢光染色分析 48
3.3.7 突觸功能性分析 49
3.4 結論 62
第四章 以聚電解質多層膜與微通道促進並導引神經網絡形成 63
4.1 研究目標 63
4.2 實驗材料、方法與儀器 63
4.2.1 材料與藥品 63
4.2.2 儀器設備 64
4.2.3 SU-8 光阻劑 66
4.2.4 光蝕刻製程 66
4.2.5 晶片組裝 69
4.2.6 神經幹細胞型態與神經球直徑統計 72
4.2.7 細胞毒性分析 72
4.2.8 神經細胞球佔比大小分析 73
4.2.9 神經軸索生長方向分析 73
4.2.10 神經網絡連接率的計算與統計 73
4.2.11 統計分析 74
4.3 結果與討論 75
4.3.1 晶片平台設計與尺寸選擇 75
4.3.2 接觸角測量 76
4.3.3 神經幹細胞細胞形態觀察 77
4.3.4 神經網絡連接與比例計算 77
4.3.5 神經網絡連接螢光染色分析 79
4.3.6 突觸功能性分析 80
4.3.7 細胞毒性分析 80
4.3.8 生長空間對於神經細胞球的影響 81
4.3.9 神經軸索生長方向導引分析 83
4.4 結論 97
第五章 神經網絡電阻抗即時分析平台 98
5.1 研究目標 98
5.2 實驗材料、方法與儀器 99
5.2.1 材料藥品 99
5.2.2 儀器設備 99
5.2.3 以光蝕刻製程製作ITO微電極 100
5.2.4 利用電阻抗分析法測量神經連接情形 103
5.2.5 單一電極電刺激裝置與電場設計 103
5.2.6 統計分析 105
5.3 結果與討論 106
5.3.1 電阻抗分析平台設計 106
5.3.2 電阻抗分析基礎值判定 106
5.3.3 神經軸索連接情況判定 107
5.3.4 神經軸索斷裂後判定 108
5.3.5 長時間連續式電阻抗值分析 108
5.3.6 單一電極電刺激對神經網絡的影響 109
5.4 結論 124
第六章 總結論 125
附錄 研究成果 127
7.1 期刊論文列表 127
7.2 國際會議參與列表 127
參考文獻 129

 
圖目錄
圖1：神經幹細胞的來源 7
圖2：神經幹細胞的(A)分化行為與(B)主要分佈區域 8
圖3：光阻材料受曝光時間影響的可能結果(A)曝光量不足；(B)曝光量過度 21
圖4：電阻抗量測二維細胞培養之基本架構 24
圖5：矽基板晶片進行電阻抗量測二維細胞培養 24
圖6：二維電極阻抗分析系統中細胞貼附、增生與死亡的阻抗分析變化圖 25
圖7：神經幹細胞分離萃取流程 35
圖8：側腦室神經幹細胞發育區辨識 35
圖9：聚電解質多層膜結構示意圖 36
圖10：電刺激系統立體結構裝置圖 42
圖11：電刺激系統結構實照 42
圖12：神經幹細胞球分化的形態圖 50
圖13：神經幹細胞經電刺激後的細胞形態 50
圖14：神經幹細胞經電刺激神經軸索長度分佈區間 51
圖15：神經幹細胞經電刺激神經軸索長度分佈區間 (區間大小：100μm) 52
圖16：神經幹細胞經電刺激神經軸索長度分佈區間 (區間大小：300μm) 52
圖17：神經幹細胞經電刺激神經軸索平均長度統計圖 53
圖18：神經幹細胞培養於不同層數的聚電解質多層膜的細胞形態 53
圖19：神經幹細胞培養於聚電解質多層膜神經軸索分佈區間 54
圖20：神經幹細胞培養於聚電解質多層膜神經軸索分佈區間 (區間大小：100μm) 54
圖21：神經幹細胞培養於聚電解質多層膜神經軸索分佈區間 (區間大小：300μm) 55
圖22：神經幹細胞培養於聚電解質多層膜神經軸索平均長度統計圖 55
圖23：神經幹細胞分別培養於聚電解質多層膜與電刺激環境神經軸索分佈區間綜合比較 (區間大小：300μm) 56
圖24：神經幹細胞培養於聚電解質多層膜與電刺激環境的LDH細胞毒性分析比較 56
圖25：神經幹細胞培養於聚電解質多層膜與電刺激環境的免疫螢光染色分析 (anti-GFAP：星狀膠細胞) 57
圖26：神經幹細胞培養於聚電解質多層膜與電刺激環境的免疫螢光染色分析 (anti-MAP2：神經元) 57
圖27：神經幹細胞培養於聚電解質多層膜與電刺激環境的免疫螢光染色分析 (anti-Nestin：中間絲蛋白) 58
圖28：神經幹細胞培養於聚電解質多層膜與電刺激環境3天後GFAP、MAP2分化比例分析 58
圖29：神經幹細胞培養於聚電解質多層膜與電刺激環境3天後MAP2、NESTIN螢光染色強度比較 59
圖30：神經幹細胞培養於聚電解質多層膜與電刺激環境3天後進行突觸功能性分析比較 60
圖31：神經幹細胞培養於聚電解質多層膜與電刺激環境3天後進行突觸功能性分析數據分析圖 61
圖32：SU-8 2050旋轉塗佈轉速與厚度對應圖 67
圖33：SU-8微通道尺寸設計圖 69
圖34：SU-8光遮罩設計圖 70
圖35：以SU-8負光阻在ITO玻璃表面製作微通道並塗佈聚電解質多層膜流程圖 71
圖36：神經網絡連接比例計算方法 74
圖37：SU-8表面的接觸角測量 85
圖38：SU-8經過氧電漿處理的接觸角測量 85
圖39：ITO材料表面接觸角量測 85
圖40：神經幹細胞細胞球培養於微通道導引晶片第5天細胞形態 86
圖41：神經幹細胞細胞球培養於含聚電解質多層膜 n=3.5的微通道導引晶片第3、5、7天的細胞形態 86
圖42：NSPCs於含有SU-8層，底部分別為ITO-Glass與ITO-PEM-n=3.5環境下培養五天後的細胞毒性分析 87
圖43：神經細胞球的直徑區間測量方式 88
圖44：經過3-5天培養後，神經細胞球的直徑區間分析 88
圖45：神經細胞球於含有SU-8微孔洞(直徑300與500 μm)中的生長空間佔比分析計算方法 89
圖46：經過3-5天培養後，神經細胞球於含有SU-8微孔洞(直徑300與500 μm)中的生長空間佔比分析 89
圖47：在SU-8孔洞直徑(A)300μm和(B)500μm中神經軸索生長方向模型 90
圖48：在直徑為300μm的SU-8微孔洞中，在沒有PEM的條件下，神經細胞球培養5天後，神經軸索分佈的長度和角度極坐標圖 90
圖49：在直徑為500μm的SU-8微孔洞中，在沒有PEM的條件下，神經細胞球培養5天後，神經軸索分佈的長度和角度極坐標圖 91
圖50：在直徑為300μm的SU-8微孔洞中，在PEM n=3.5條件下，神經細胞球培養5天後，神經軸索分佈的長度和角度極坐標圖 91
圖51：在直徑為500μm的SU-8微孔洞中，在PEM n=3.5條件下，神經細胞球培養5天後，神經軸索分佈的長度和角度極坐標圖 92
圖52：神經細胞球培養5天後，在直徑為300μm與500μm的SU-8微孔洞中，在含/不含PEM n=3.5條件下，神經軸索的分佈區間圖 92
圖53：神經幹細胞細胞球於含PEM n=3.5的微通道導引晶片培養第五天神經網絡已成功連結 93
圖54：同位置神經幹細胞細胞球於含PEM n=3.5的微通道導引晶片培養第3天與第5天神經網絡連結率計算 94
圖55：神經網絡連接比例分析統計圖 95
圖56：神經細胞球在聚電解質多層膜n=3.5條件下培養5天後，神經網絡連接螢光染色分析 96
圖57：突觸功能性分析 96
圖58：ITO微電極設計圖 101
圖59：ITO微電極(A)單一區域設計圖，ITO表面具有500μm的細胞檢測區域，電極線寬150μm與(B)製作流程 102
圖60：單一電極電刺激裝置構造與電場圖 104
圖61：電刺激電極階位計算示意圖 105
圖62：ITO電極實際製作圖與尺寸量測分析 113
圖63：神經網絡阻抗分析設計-分層解析圖 114
圖64：神經網絡阻抗分析設計-側面圖 114
圖65：神經網絡阻抗分析設計-俯視圖 115
圖66：神經網絡阻抗分析設計-結構分解圖 115
圖67：神經細胞球在具有聚電解質多層膜(n=3.5)的ITO微電極和SU-8負光阻微通道表面的製備示意圖(未按比例) 116
圖68：電阻抗分析檢測路徑 117
圖69：相鄰兩神經細胞球連接/未連接的電阻抗關係 117
圖70：相鄰神經細胞球之間在不同條件下透過神經軸索連接/未連接的電阻抗值關係 118
圖71：神經細胞球在聚電解質多層膜與SU-8微通道培養5天後(A)全區域神經細胞球皆連接與(B)電阻抗值分析 (孔洞直徑為500μm，通道長度為400μm) 119
圖72：神經細胞球在聚電解質多層膜與SU-8微通道培養5天後(A)部分區域神經細胞球連接與(B)電阻抗值分析 (孔洞直徑為500μm，通道長度為600μm) 120
圖73：細胞經過三天培養神經軸索相連時的阻抗值分析值與第五天神經軸索斷裂後的阻抗分析值比較 121
圖74：長時間連續式電阻抗值數據分析圖 122
圖75：經單一電極電刺激之神經幹細胞球(A)(位置：A1)與周邊未經電刺激之神經幹細胞球(B)(位置：C3) 122
圖76：單一電極電刺激神經幹細胞球形成神經網絡的連結比例分析 123

表目錄
表 1：Tris buffer 配製材料...................................................................26
表 2：Neural Stem Cell Culture Medium..............................................27
表 3：1X PBS Solution .........................................................................27
表 4：Contents of HBSS Solution.........................................................28
表 5：Contents of High Potassium HBSS Solution ...............................28
表 6：Contents of FM1-43 Lipid Dye Solution .....................................29
表 7：Contents of 10X EDTA PBS Solution .........................................29
表 8：Contents of 0.05% Trypsin Solution............................................29
表 9：聚電解質多層膜組裝過程.........................................................33
表 10：用於神經細胞免疫螢光染色的抗體列表................................39
表 11：ITO 表面塗佈 SU-8 之光蝕刻製程步驟..................................68
表 12：ITO 微電極製備步驟 .............................................................102

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