在人體血液過濾當中，腎小球扮演著十分重要的角色。腎小球由內而外分別是內皮細胞、腎小球基底膜和腎足細胞。三者分別扮演著不同孔洞大小的過濾膜來過濾血液中需要留下來的東西，像是內皮細胞不讓紅血球通過、腎足細胞限制了血紅素或是白蛋白的流出。因此我們必須做出管狀且多層共軸結構，成功的讓管子由內而外培養內皮細胞、腎小球基底膜和腎足細胞，才能模仿人體內腎小球的行為原理。過去的研究當中，要做成長條形的管狀共軸結構，外層都是用水膠，利用水膠碰到氯化鈣水溶液，水溶液當中的鈣離子會置換海藻酸鈉中的鈉離子產生交聯作用而固化來維持管狀的機械強度，內層通常都是以明膠混合著所要培養的細胞，內層外層同時用注射式幫浦注射而做出共軸雙層的結構。在第一階段的研究當中，我們致力於壓力對於腎足細胞的平面影響，畢竟台灣洗腎人口居高不下，洗腎密度高居世界前五，而高血壓又是洗腎的主要原因之一，因此先行在平面探討壓力對腎足細胞過濾效果的病理影響。有了第一階段的病理結果，在第二階段，我們重點放在尋找三維管狀結構的各種參數，包含製造三維管子的各種流速、材料種類、材料濃度等等。畢竟細胞在三維立體結構中，細胞的生長情況本來就不如在平面結構，而且在管中，細胞的生長環境不只有培養液，還有水膠以及交聯用的二價金屬離子，這些對細胞的生長都存在著影響。因此本階段就是在二維環境中尋找最佳參數以運用到三維管子的製造。至於最後一個階段則是實際製造出三維的管子。在實驗中發現腎足細胞不喜歡有氯化鈣的環境，因此我們進一步尋找其他二價離子使水膠固化。發現鍶離子不但對腎足細胞無害，能使腎足細胞進行分裂的行為，這一個步驟是腎小球內過濾的關鍵因素，除此之外，氯化鍶能讓水膠有更好的機械強度，這也為實驗未來接管的部分找到更佳的材料。
目前先以單一細胞去做實驗，未來的目標是做出三層結構以模仿腎小球內部的構造，而且內層內皮細胞以及外層腎足細胞所需要的生長環境也不太相同，因此需要在兩者之間取一平衡點讓兩者都能順利生長的最佳環境。

Glomerular play an important role of blood filtration in human body。From outside to inside are endothelia cells, glomerular basement membrane(GBM) and podocyte, respectively. These three kinds of cells act as the different size of filtration membranes to filter. For example, endothelia cells stop red blood cells crossing the blood vessel. Therefore we have to manufacture a core-shell multilayer co-axis microfibers, and culture three kinds of cell on the each layer. So that we can imitate the behavior of glomerular in human boddy.
In the past research, alginate is the only material of the co-axis microfibers. Alginate will have a cross-linking reaction when we add calcium chloride solution in it. The sodium ion in alginate will be replaced by the calcium ion. The hard alginate have to hold the microfibers by its mechanical strength. The material of the inside tube is gelatin with suspended cells. We use two syringe pump to produce the outside tube and inside tube at the same time. In the first step of our experiment, we focus on the influence of podocyte after giving pressure differences. The dialysis population still high is Taiwan. The density of dialysis population ranks in top five in all over the world. And hypertension is one of the leading causes of dialysis. So we do some research on podocyte filtration after giving pressure. With the pathological behavior and results in first step, we find out some best parameters including gel inject velocity, gel species, gel concentration in second step. Cells is not easily to survive in 3D structures. In 3D structures, there are many impact factors. They will influence the survival ratio of cells. In this step, we focus on finding the best environment for cells to survive. And supply these parameters to 3D structure cells culturing. In the last step, we manufacture 3D cell tube. In this period, we observe that podocyte are sensitive to calcium ion. So we cure alginate with strontium, instead. Podocyte stretch out under the environment of strontium. It is the key factor of glomerular filtration. Besides strontium cured alginate has a more stronger mechanical strength. This is the right material for the 3D cell tube.
For the future work, multi-layer 3D cell tube with endothelial cell, GBM and podocyte is our final purpose to mimic the glomerulr in vitro.

摘要 ii
Abstract iv
致謝 vi
圖目錄 ix
表目錄 xii
第一章 緒論 1
1.1研究背景 1
1.2研究動機 2
1.3研究目的 4
第二章 文獻回顧 7
2.1組織工程 7
2.1.1細胞貼附 8
2.2水膠 10
2.3膠原蛋白(collagen) 14
2.4二維空間水膠培養細胞 16
2.5三維空間水膠培養細胞 17
第三章 實驗設計與規劃 22
3.1平面壓力測試 23
3.1.1 發病機理 23
3.1.2 平面壓力實驗設計 24
3.2 二維水膠環境模擬 25
3.2.1 二維模擬動機 25
3.2.2 二維細胞培養設計 28
3.3三維細胞管製作 29
3.3.1 裝置改善 29
第四章 實驗操作流程 31
4.1細胞微環境操作 31
4.1.1 細胞培養基本技術 31
4.1.2調配培養液 32
4.1.3 細胞培養 32
4.2平面壓力測試實驗流程 33
4.2.1膜上培養細胞 33
4.2.2進行壓力測試 34
4.2.3壓力測試後分析 35
4.3二維水膠培養流程 36
4.3.1水膠製備 36
4.3.2 氯化鈣、氯化鎂、氯化鍶測試 36
4.2.3 塗佈膠原蛋白 37
4.2.4 塗佈膠原蛋白 38
4.4實驗儀器 39
4.4.1膜上培養細胞儀器與藥品 39
4.4.2水膠培養細胞儀器與藥品 42
4.4.3三維細胞管儀器與藥品 44
第五章 結果與討論 47
5.1平面壓力實驗結果 47
5.1.1蛋白質洩漏測試 47
5.1.2 mRNA表現量測試 48
5.1.3 恢復實驗 49
5.2二維水膠細胞培養結果 51
5.2.1各類參數影響比較 51
5.2.2不同水膠濃度細胞培養 54
5.3三維細胞管培養結果 55
5.3.1各類細胞管內培養 55
第六章 結論 59
6.1平面壓力測試 59
6.2二維水膠表面細胞培養 60
6.2.1各類參數的影響 60
6.2.2不同濃度水膠培養 62
6.3三維細胞管製作 63
6.3.1各種細胞管內培養 63
第七章 未來工作 64
第八章 文獻回顧 66

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