近年來，組織工程學以回復受損器官之功能為重要研究方向，並基於組織工程學原理利用自組裝的治療策略，也就是利用細胞本身固有之分泌細胞外基質以及細胞激素能力來建立一個適合植入受損區域的細胞移植物。但是，在固有的細胞培養環境中，培養基為一過度稀釋的微環境，需要耗費許多時間才得以使細胞分泌出足夠豐富去治療受損區域的細胞外基質。先前已有許多文獻指出，間葉幹細胞本身能夠分泌大量的細胞外基質以及細胞激素，並且在培養時利用巨分子擁擠現象原理，即便在低血清濃度的培養基微環境下，亦可模擬體內較為擁擠之培養環境，便可以有效提升二維細胞的細胞外基質沉積量並影響細胞外基質的型態；另有研究指出在培養間葉幹細胞時加入抗壞血酸(Ascorbic acid)能夠提升膠原蛋白的分泌量。然而，即便利用巨分子擁擠現象或是在培養時加入抗壞血酸，要產生出足夠豐富的細胞外基質量仍須花費一定的時間。又先前本實驗室有研究指出製備三維幹細胞球體相較於傳統二維培養幹細胞能夠縮短培養時間並且分泌更多的細胞外基質以及更多的促神經生長、促存活及血管新生因子以達到更好的治療效果。為更有效提升三維幹細胞球之治療潛能，本研究使用甲基纖維素水膠系統使間葉幹細胞自我貼附並沉降細胞外基質，並綜合本實驗室先前之實驗中所證實Ficoll 400 做為巨分子擁擠分子時能後提升細胞外基質的分泌量結果，於培養過程中加入Ficoll 400 做為巨分子並添加抗壞血酸，分析不同條件增加間葉幹細胞之細胞外基質沉降速度、細胞激素含量以及免疫調節能力，優化培養條件以達到短時間建立可應用於臨床治療之富含細胞外基質及細胞激素之三維幹細胞球體。體外實驗中，我們以醣胺聚糖定量分析、膠原蛋白定量分析以及酵素連結免疫吸附分析法等方式確認培養基內有巨分子以及抗壞血酸的存在確實能夠影響細胞外基質及細胞激素的沉積量及分泌量；並以即時聚合酶連鎖反應以及免疫螢光染色等方式確認不同培養條件之三維間葉幹細胞球體之免疫調節能力；最後在體內實驗則以冷光訊號偵測檢驗不同培養貼件之三維幹細胞球體之細胞存活能力。希望可以藉由優化最適當的三維幹細胞球培養環境，以達到縮短細胞治療時之準備時間，提升三維幹細胞球之細胞治療潛力並應用於受損區域。

In recent years, the most important research goal in tissue engineering is recover the function of injured organs. Based on the self-assembly principal of tissue engineering, which is, using the extracellular matrix (ECM) and cytokines excreted by cells, to construct a transplant suitable for embedding into the injured area. However, in normal culture environment, the media are diluted microenvironment, obstructing cells to excrete sufficient ECM to cure the injured area. According to studies, mesenchymal stem cells (MSC) itself can not only secrete abundant ECM and cytokines. In addition, by utilizing macromolecular crowding, even in the low-serum media can simulate the crowded culture in vivo. In this way, we can efficiently increase the 2-dimensional(2D) ECM deposition and effect the morphology of the ECM. Other studies showed that by adding ascorbic acid can promote the deposition of collagen. However, even by utilizing macromolecular crowding or adding ascorbic acid in the media, it still takes long time to excrete sufficient ECM. Additionally, our previous research show that we can shorten the culture time by using 3-dimensional (3D) MSC spheroid and allow it to secrete more ECM and other pro-neurogenic and pro-angiogenic potentials comparing to 2D culture. In order to promote the therapeutic potential of 3D MSC spheroid, our study uses self adhesion and deposition of ECM in our methylcellulose hydrogel system. Combining our previous studies, which proved the Ficoll 400 can be used to promote ECM deposition in macromolecular crowding, we add Ficoll 400 as macromolecules and ascorbic acid to optimize the different parameters that accelerate MSC to deposit ECM, cytokines, and immuno-modulation ability. In in vitro studies, we quantify the glycosaminoglycan, collagen and growth factors to confirm that the macromolecules and ascorbic acid added in medium can affect the ECM deposition and cytokine secretion. Besides, we use real-time PCR and immunofluorescence staining to analyze the different ways in immuno-modulation. Finally, we use luminescence signal to test the viability in in vivo studies, in order to shorten the cell preparation time and promote therapeutic potential at the injured site by optimizing the best culture environment.

摘要 ....................................................................................................................................................... I Abstract .............................................................................................................................................. II
目錄 .................................................................................................................................................... III
圖目錄 ................................................................................................................................................ VI
表目錄 ................................................................................................................................................ IX
第一章、緒論 ...................................................................................................................................... 1
1-1、前言 ............................................................................................................................................ 1
1-2、細胞外基質 (Extracellular matrix, ECM) .............................................................................. 2
1-2-1、結構性蛋白質 (Structural protein) ................................................................................. 3
1-2-2、非結構性蛋白質 (Non-structural protein) ...................................................................... 3
1-2-3、蛋白聚醣 (Proteoglycans) ............................................................................................... 4
1-2-4、生長因子 (Growth factor) ............................................................................................... 4
1-2-5、基質金屬蛋白酶 (Matrix metalloproteinases, MMPs) .................................................. 4
1-3、巨分子擁擠現象 (Macromolecular crowding, MMC) ........................................................... 5
1-3-1、Dextran sulfate 500 kDa (DxS) ....................................................................................... 8
1-3-2、Ficoll 400 kDa (Fc400) ................................................................................................... 8
1-3-3、Cocktail of Ficoll 70 kDa (Fc70) and Ficoll 400 kDa ..................................................... 9
1-3-4、文獻回顧 ....................................................................................................................... 10
1-4、抗壞血酸 (L-Ascorbic acid, AA) ............................................................................................ 10
1-5、間葉幹細胞 (Mesenchymal stem cell, MSC) ........................................................................ 13
1-5-1、旁分泌作用 (Paracrine effects)..................................................................................... 15
1-5-2、免疫調節能力 (Immuno-modulation) .......................................................................... 16
1-5-3、細胞療法之應用 ........................................................................................................... 20
1-6、小鼠神經微膠質細胞 (Mouse microglial cell, BV2) ............................................................. 21
1-6-1、神經微膠質細胞的極化 ............................................................................................... 21
1-6-2、間葉幹細胞之細胞療法於發炎反應之應用 ............................................................... 23
1-7、甲基纖維素 (Methylcellulose, MC) ....................................................................................... 23
1-8、三維幹細胞球體於細胞療法的效用 ...................................................................................... 25
1-9、研究動機與實驗目的 .............................................................................................................. 27
第二章、材料和方法 ........................................................................................................................ 30
2-1、細胞培養 .................................................................................................................................. 30
2-2、甲基纖維素水膠製備 .............................................................................................................. 31
2-3、三維幹細胞球體製備 .............................................................................................................. 31
2-4、三維幹細胞球體型態分析 ...................................................................................................... 32
2-5、條件培養液 (Conditioned medium, CM) 製備 ..................................................................... 32
2-6、體外發炎反應模型建立 .......................................................................................................... 33
2-7、細胞免疫螢光染色 .................................................................................................................. 33
2-7-1、二維細胞免疫螢光染色 ............................................................................................... 33
2-7-2、三維幹細胞球體免疫螢光染色 ................................................................................... 33
2-8、醣胺聚醣定量分析 (Glycosaminoglycan assay) ................................................................... 34
2-9、天狼星紅快速綠色膠原蛋白染色分析 (Sirius red/fast green collagen staining assay) .... 35
2-10、管狀形成分析 (Tube formation assay)................................................................................ 35
2-11、酵素連結免疫吸附分析法 (Enzyme-linked immunosorbent assay, ELISA) ................... 36
2-12、即時聚合酶連鎖反應 (Real-time polymerase chain reaction, Real-time PCR) .............. 37
2-13、動物模式 (In vivo study) ....................................................................................................... 42
2-14、統計分析 ................................................................................................................................ 42
第三章、實驗結果與討論 ................................................................................................................ 43
3-1、三維幹細胞球體型態和特性 .................................................................................................. 43
3-2、加入巨分子擁擠分子後三維幹細胞球體的細胞活性及抗細胞凋亡能力 .......................... 45
3-3、加入巨分子擁擠分子後三維幹細胞球分泌細胞外基質之能力 .......................................... 46
3-3-1、細胞外基質 (Extracellular matrix) 分布 ...................................................................... 46
3-3-2、醣胺聚醣定量分析 ....................................................................................................... 47
3-3-3、天狼星紅快速綠色膠原蛋白染色分析 ....................................................................... 47
3-3-4、即時聚合酶連鎖反應 ................................................................................................... 48
3-4、加入巨分子擁擠分子後三維幹細胞球分泌生長因子之能力 .............................................. 48
3-4-1、即時聚合酶連鎖反應 ................................................................................................... 49
3-4-2、人類血管內皮生長因子-A 酵素連結免疫吸附分析 .................................................. 49
3-5、加入巨分子擁擠分子後三維幹細胞球體的促血管新生能力 .............................................. 50
3-5-1、人類血管內皮生長因子-A 酵素連結免疫吸附分析 .................................................. 50
3-5-2、管狀形成分析 ............................................................................................................... 51
3-6、加入巨分子擁擠分子後三維幹細胞球體的免疫調節能力 .................................................. 52
3-6-1、即時聚合酶連鎖反應 ................................................................................................... 53
3-6-2、酵素連結免疫吸附分析法 ........................................................................................... 54
3-7、三維幹細胞球體調節體外發炎模型能力 .............................................................................. 56
3-7-1、免疫螢光染色 ............................................................................................................... 56
3-7-2、即時聚合酶連鎖反應 ................................................................................................... 59
3-8、動物實驗 .................................................................................................................................. 60
第四章、討論與結論 ........................................................................................................................ 63
參考文獻 ............................................................................................................................................ 64

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