膠質母細胞瘤(Glioblastoma, GBM)是非常難以治療的癌症，化學治療受限於血腦屏障使藥物無法有效遞送，手術切除過程為了保有大腦原有功能導致腫瘤難以根除。仿生腦瘤體外模型的開發將有助於腫瘤與周邊細胞關係的探討並能提供更貼近體內環境的治療測試結果。
本研究合成以甲基丙烯酸縮水甘油酯(Glycidyl methacrylate, GMA)改質蠶絲蛋白形成甲基丙烯酸縮水甘油酯改質蠶絲蛋白(Silk-GMA)並結合以甲基丙烯酸酐(Methacrylic anhydride, MA)改質透明質酸(Hyaluronic acid, HA)的甲基丙烯酸化透明質酸(HAMA)，製作出2種生物墨水並進行混合，目的要以3D生物列印製作出3D腦瘤體外模型(in vitro Glioblastoma Model)
首先以傅立葉轉換紅外光譜(Fourier-transform infrared spectroscopy, FTIR)與核磁共振光譜儀(1H-NMR)分析GMA和MA對蠶絲蛋白與透明質酸的成功改質與修飾程度。
在過去研究已發現在腦瘤進展生長的過程中腫瘤微環境基質硬度會有較硬的情形，因此將Silk-GMA添加不同濃度HAMA製備出系列不同基質濃度組成的水膠，並藉由楊氏模量(Young's modulus)判斷系列水膠的軟硬程度。結果顯示隨著HAMA濃度的增加楊氏模量也隨之提升，而本研究中8wt%Silk-GMA/3wt%HAMA水膠的楊氏模量與文獻中老鼠腦瘤組織硬度範圍近似，更增加了腦瘤微環境的模擬程度。流變(Rheology)測試也發現系統中添加HAMA濃度越高，儲能模量(G’)、損耗模量(G’’)和黏度皆提升；在掃描式電子顯微鏡(Scanning electron microscopy, SEM)影像中顯示水膠微結構呈現適合細胞生長的多孔結構，隨著添加HAMA濃度越高，孔洞會越小越緻密；在可列印性測試中也發現，添加較高HAMA的生物墨水組別也有較高的可列印能力，這提升了細胞包埋列印的可控制性。細胞生物適合性測試顯示系列Silk-GMA/HAMA水膠皆表現出優異的生物相容性，顯見Silk-GMA/HAMA生醫材料適合使用於組織工程與體外模型建構。在抗癌藥物試驗中，三維神經膠質瘤球體懸浮培養與傳統二維貼附型培養相比，三維系統表現更高的IC50，代表三維腫瘤球結構具有較高抗藥性，此結果也顯示三維系統更貼近真實腫瘤環境。
為了提升腫瘤球的擬真度，本研究製備巨噬細胞與腦瘤細胞的異質球體(Heterospheroid)系統，並以活體細胞染劑Cell tracker進行觀察。結果顯示以不同比例進行混合培養後，異質球體中腦瘤細胞占比均較巨噬細胞高，並利用Cell tracker在細胞遷移實驗中，發現細胞球體在添加免疫細胞以及較硬的Silk-GMA/HAMA生物墨水中都呈現較高的遷移力。
最後為了探討Silk-GMA/HAMA生物墨水對於建立腦瘤體外模型的可行性，並探討三種基質組成對腫瘤微環境的影響，將異質球體包埋於Silk-GMA/HAMA生物墨水中並以擠出式進行3D生物列印，分別觀察巨噬細胞M1及M2標記物以及膠質母細胞瘤特異性標記物。結果顯示在HAMA添加量最高的組別-8wt%Silk-GMA/3wt%HAMA生物墨水組別，M2巨噬細胞標記物呈現最高的表達，顯見此微環境有助於促進巨噬細胞轉變為抑制發炎的M2型並進而促進腫瘤生長與侵犯行為。而膠質母細胞瘤特異標記物表達在三個組別中較無明顯差異。與2D貼附培養和3D懸浮培養相比，包埋於生物墨水中較能明顯展現出2種特異標記物的高表達。本研究顯見可利用不同生物墨水的基質組成與軟硬度變化與複合腫瘤球體的包埋列印成功建立3D 膠質母細胞瘤體外模型，並可模擬腫瘤相關巨噬細胞(Tumor associated macrophages, TAMs)在不同期程的膠質母細胞瘤中的行為表現與異質球體遷移力的影響。
本篇研究以Silk-GMA/HAMA生物墨水3D多細胞列印建構出膠質母細胞瘤體外模型，成功促進M2巨噬細胞的極化以及腦瘤細胞的生長，未來希望能夠列印出更複雜圖案並加入更多細胞來觀察彼此間的作用關係，打造出更仿生的腦瘤體外模型，或是建立其他疾病模型，並與體內(in vivo)實驗做比較，增加體外模型的可應用性。

The effectiveness of chemotherapy in treating brain tumors is often limited by the blood-brain barrier, which hampers efficient drug delivery. Additionally, surgical removal procedures aim to preserve brain functions, making it difficult to completely eradicate tumors. To address these challenges, researchers have developed biomimetic in vitro brain tumor models that can better investigate tumor interactions with surrounding cells and provide treatment testing results that closely resemble the in vivo environment.
In this study, silk fibroin (SF) was modified with glycidyl methacrylate (GMA) to synthesize Silk-GMA, while hyaluronic acid (HA) was modified with methacrylic anhydride (MA) to form methacrylate HA (HAMA). Different composition of Silk-GMA and HAMA were used to prepare series of bioinks for 3D bioprinting, with the goal of constructing a 3D glioblastoma (GBM) in vitro model.
The modifications and functionalization of SF and HA with GMA and MA were analyzed using Fourier-Transform Infrared Spectroscopy (FTIR) and 1H Nuclear Magnetic Resonance (1H-NMR) spectroscopy, respectively. Previous studies have demonstrated that the matrix stiffness in tumor microenvironment increased during tumor progression. In this study, a series of hydrogels with different stiffness were prepared by incorporating various concentrations of HAMA into Silk-GMA. The stiffness of the hydrogels was determined by measuring the Young's modulus. As the concentration of HAMA increased, the Young's modulus also increased. The results demonstrated that the hydrogel with a composition of 8wt% Silk-GMA/3wt% HAMA exhibited a Young's modulus within the range of mouse brain tumor tissue stiffness, thereby better simulating the brain tumor microenvironment.
Rheological analysis showed that higher concentrations of HAMA led to increased storage modulus (G'), loss modulus (G''), and viscosity of the hydrogels. Scanning electron microscopy (SEM) images revealed that the hydrogel exhibited a porous microstructure suitable for cell growth. As the concentration of HAMA increased, the pore size decreased, resulting in a denser structure. Furthermore, the bioink with higher HAMA content demonstrated printability improvement, enhancing the controllability of cell laden 3D bioprinting. In addition, cell toxicity assays demonstrated that series of Silk-GMA/HAMA hydrogels prepared this study exhibited high biocompatibility.
Anti-cancer drug resistance test revealed that the 3D GBM spheroids showed higher IC50 values compared to traditional 2D adherent cultures. It is considered that 3D tumor spheroid system better mimics the real tumor environment.
To enhance the authenticity of tumor spheroids, heterospheroids were formed by co-culturing macrophages with GBM cells. Cell tracker staining revealed that GBM cells dominated the heterospheroids in all mixed ratios. Cell migration test demonstrated that the cell spheroids exhibited higher migratory ability when immune cells and stiffer Silk-GMA/HAMA bioink were present.
To investigate the feasibility of using Silk-GMA/HAMA bioink for constructing a GBM in vitro model and assess the impact of different matrix stiffness compositions on the tumor microenvironment, heterospheroids were embedded in Silk-GMA/HAMA bioink and 3D bioprinted using an extrusion-based method. The expression of M1 and M2 macrophage markers, as well as GBM-specific markers, was observed. The bioink with the highest HAMA concentration (8wt% Silk-GMA/3wt% HAMA) exhibited the highest expression of the M2 macrophage marker. This indicates a microenvironment that promotes macrophage polarization into the anti-inflammatory M2 type and enhances tumor growth and invasion. However, the expression of GBM-specific markers did not significantly differ among the three groups. Notably, embedding in the bioink showed higher expression of the two specific markers compared to 2D adherent and 3D suspension cultures.
This study successfully established a 3D GBM in vitro model using Silk-GMA/HAMA bioink with varying degrees of stiffness. The model accurately simulated the behavior of tumor-associated macrophages (TAMs) in GBM at different stages and the impact of heterospheroids' migratory ability. In the future, more complex and biomimetic patterns will be designed and additional cell types will also be included to observe different types of cell-cell interactions and to create a more biomimetic brain tumor in vitro model. Additionally, establishing models for other diseases and comparing them with in vivo experiments will enhance the applicability of in vitro models.

致謝 i
中文摘要 ii
Abstract v
目錄 ix
圖目錄 xiii
表目錄 xvii
第一章 緒論 1
1.1研究動機與目的 1
1.2實驗架構 4
第二章 文獻回顧 5
2.1大腦神經膠質母細胞瘤 5
2.2 免疫抑制腫瘤微環境 5
2.3大腦基質對腦瘤的影響 7
2.4生物墨水 10
2.4.1生物墨水種類 11
2.4.2 蠶絲蛋白及其應用 11
2.4.3透明質酸及其應用 16
2.4.4生物墨水中的高分子交聯機制 21
2.4.4.1物理交聯 21
2.4.4.2化學交聯 22
2.5 3D列印 23
2.5.1 3D列印方法 24
2.6大腦腫瘤體外模型 25
2.6.1腦瘤晶片 25
2.6.2腦瘤類器官 27
2.6.3 3D列印在腦瘤體外模型上的應用 30
第三章 實驗材料與方法 39
3.1藥品及材料 39
3.2儀器 42
3.3細胞培養 44
3.3.1不同細胞培養基配製 44
3.3.2 磷酸鹽緩衝溶液(Phosphate buffered saline, PBS )配製 44
3.3.3 L929細胞培養與繼代 45
3.3.4 GL261細胞培養與繼代 45
3.3.5 RAW264.7細胞培養與繼代 46
3.3.6細胞計數 47
3.4材料合成 47
3.4.1甲基丙烯酸縮水甘油酯改質蠶絲蛋白(Silk-GMA)的製備 47
3.4.2 甲基丙烯酸化透明質酸(HAMA)的製備 49
3.5生物墨水以及水膠合成 50
3.5.1 Silk-GMA/HAMA生物墨水製備 50
3.5.2 PDMS水膠模具製備 51
3.5.3 Silk-GMA/HAMA水膠製備 51
3.6 Silk-GMA/HAMA性質測試 51
3.6.1 核磁共振光譜儀 51
3.6.2傅立葉轉換紅外光譜 52
3.6.3壓縮模量 52
3.6.4膨潤度 52
3.6.5 掃描式電子顯微鏡 52
3.6.6流變性質 53
3.6.6.1頻率掃描 53
3.6.6.2應變掃描 53
3.6.6.3剪切稀化 53
3.6.7可列印性測試 53
3.7 Silk-GMA/HAMA水膠生物相容性測試 54
3.7.1 MTT測試 54
3.7.2 LDH 測試 55
3.7.3 Live/Dead測試 55
3.8 3D腦瘤細胞球體的製備 56
3.8.1 聚乙烯醇(Polyvinyl alcohol) 56
3.9 Temozolomide(TMZ)化療藥物測試 57
3.9.1 2D貼附細胞的藥物測試 57
3.9.2 3D腫瘤球體的藥物測試 58
3.10 3D神經膠質瘤異質球體 59
3.10.1 3D神經膠質瘤異質球體製備及共培養 59
3.10.2以Cell tracker 輔助3D異質球體細胞的連續觀察 60
3.10.3 3D神經膠質瘤異質球體於貼附環境遷移情形 62
3.11 3D異質細胞球體的包埋列印 62
3.11.1 3D神經膠質瘤異質球體於不同透明質酸濃度生物墨水生長差別 62
3.12 共培養巨噬細胞極化測定 64
3.12.1 LPS/IL-4巨噬細胞極化測定 64
3.12.2 2D貼附共培養巨噬細胞極化測定 67
3.12.3 3D神經膠質瘤異質球體巨噬細胞極化測定 68
3.12.4 3D神經膠質瘤異質球體包埋列印於不同透明質酸濃度生物墨水巨噬細胞極化測定 70
3.13 共培養腦瘤細胞標記物測定 72
3.13.1 2D貼附共培養腦瘤細胞標記物測定 72
3.13.2 3D神經膠質瘤異質球體腦瘤細胞標記物測定 74
3.13.3 3D異質細胞球體包埋列印於不同透明質酸濃度生物墨水腦瘤細胞標記物測定 76
3.14統計方法 78
第四章 結果與討論 79
4.1 Silk-GMA與HAMA材料合成及鑑定分析 79
4.1.1 Silk-GMA於1H-NMR取代度分析 79
4.1.2 HAMA於1H-NMR取代度分析 80
4.1.3 Silk-GMA於FTIR分析 82
4.1.4 HAMA於FTIR分析 83
4.2 Silk-GMA/HAMA水膠合成結果 85
4.3 Silk-GMA/HAMA材料性質 88
4.3.1Silk-GMA/HAMA水膠壓縮模量測試結果 88
4.3.2 Silk-GMA/HAMA水膠膨潤度測試結果 89
4.3.3 Silk-GMA/HAMA水膠掃描式電子顯微鏡結果 90
4.3.4 Silk-GMA/HAMA水膠流變測試結果 92
4.3.5 Silk-GMA/HAMA生物墨水可列印性測試結果 96
4.4 Silk-GMA/HAMA水膠生物相容性測試結果 97
4.5 3D腦瘤細胞球體製備 101
4.6 比較二維培養與三維腫瘤球的化療藥物測試結果 105
4.7 3D神經膠質瘤異質球體製備 108
4.8 改變生物墨水透明質酸濃度於包埋列印3D異質腫瘤球的遷移影響 111
4.9 改變生物墨水透明質酸濃度於共培養多細胞腫瘤球中巨噬細胞極化以及腦瘤標記物的測試結果 114
4.10 Silk-GMA/HAMA生物墨水擠出式3D生物列印表現 134
第五章 結論 138
第六章 參考文獻 140

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