在人體循環系統當中，腎臟扮演著十分重要的角色，主要負責過濾代謝廢物以及再吸收有用的養分。而腎臟中，約由200萬個腎元組成，每一個腎元是由腎小體及腎小管所組成。而腎小球，腎小體中用於將血液過濾的組織，由內而外分別是內皮細胞、腎小球基底膜和腎足細胞。此三者細胞有著不同的孔洞大小，可以用來過濾血液中不同的物質，例如內皮細胞可擋住紅血球，但能讓血漿與水溶性蛋白通過，而腎足細胞限制血紅素及蛋白的流出。
因此，為了模擬人體中腎小球的構造，我們必須做出多層細胞管狀結構，使我們能研究其行為原理，進一步來發展仿生腎臟組織。在我們的實驗中，我們利用海藻酸鈉來當作細胞貼附的基質，利用海藻酸鈉碰到二價離子時，置換海藻酸鈉中的鈉離子，產生交聯作用固化來維持機械強度，再利用同軸針及注射式幫浦做出中空水膠管，進而在上面培養我們所想要的細胞。
第一階段研究中，我們將塗佈膠原蛋白但卻未培養細胞的氧化鋁薄膜進行加壓測試，並與培養腎足細胞的薄膜進行比較，探討在不同壓力下，腎足細胞在平面裝置的過濾能力。第二階段的研究，主要尋找混合細胞培養的各種參數，包含培養液與溫度兩大條件，因最後想要做出多層細胞的結構，所以細胞可能在非最佳環境中成長，因此必須先行測試不同生長環境對於細胞生長速率的影響，試著找出能讓兩種細胞都順利成長的環境。至於最後一階段則是實際製造出三維細胞水膠管，主要利用海藻酸鈉做為管子基材，利用鍶離子使海藻酸鈉固化，在管上進行表面處理後培養細胞，再用影像記錄其生長狀況。

In the human circulatory system, the kidneys play a very important role, mainly responsible for the filtration of metabolic waste and the reabsorption of useful nutrients. In the kidneys, about 2 million of the nephron, each nephron is composed of renal corpuscle and renal tubule. The glomerulus, the renal corpuscle uses in the blood filtration organization, from the inside and outside is the endothelial cell, the glomerular basement membrane and the podocytes. These three cells have different pore sizes that can be used to filter different substances in the bloodstream, such as endothelial cells that block red blood cells, but allow the plasma and water-soluble proteins to pass through, and podocytes limit the heme.
Therefore, in order to simulate the structure of human glomerular, we should make the multilayer cell tubular structure, so that we can study its behavior principle, further develop the bionic kidney tissue. In our experiment, sodium alginate was used as substrate for cell attachment. When sodium alginate is encountered with bivalent ions, the sodium ions in sodium alginate are replaced by the crosslinking action to maintain the mechanical strength, then the coaxial needle and syringe pump are used to make the hollow water hose, and then we can culture cells which we want.
In the first stage of the study, we tested the AAO membrane coated with collagen but not cultured cells, and compared it with the membrane of the cultured podocytes to discuss the filtration ability of the podocytes under different pressures. The second stage of the study, mainly in the search for co-culture of different cells, including the two conditions of medium and temperature, because the last want to make the structure of multilayer cells, so the cell may grow in a nonoptimal environment. It is necessary to test different environments for the growth rate of cells, and try to find the environment that allows both cells to grow smoothly. The final stage, it is the manufacture of three-dimensional and multi-layer coaxial tube with cells culture, mainly using sodium alginate as a base material, and use strontium ions to cure sodium alginate. Then making surface treatment on alginate tube, and using image to record the cells’ growth condition.

摘要 ii
Abstract iii
致謝 v
總目錄 vi
表目錄 viii
圖目錄 ix
第一章 序論 1
1.1前言 1
1.2研究動機 2
1.3研究目的 3
第二章 文獻回顧 6
2.1影響腎足細胞過濾因素 6
2.2腎足細胞與腎臟疾病關係 8
2.3組織工程 9
2.3.1 生物材料 14
2.4水膠 16
2.4.1 海藻酸鈉（Sodium alginate）的來源與性質 21
2.4.2 不同離子對海藻酸鈉的固化影響 22
2.4.3 明膠（Gelatin）的來源與性質 23
2.5細胞貼附 23
2.6水膠表面培養細胞 25
2.6.1 三維空間水膠培養細胞 26
第三章 實驗設計與規劃 28
3.1平面壓力測試設計 28
3.1.1 發病機理 28
3.1.2 平面壓力實驗 28
3.2 混合細胞培養測試 30
3.2.1 混合細胞培養動機 30
3.2.2 混合細胞培養設計 30
3.3三維多層細胞管製作 30
3.3.1 三維單層細胞管改良 30
3.3.2 三維多層細胞管實驗設計 32
3.3.3 三維細胞管冷凍保存設計 33
第四章 實驗操作流程 34
4.1微生物實驗室基本操作 34
4.1.1細胞培養基本技術 34
4.1.2調配培養液 34
4.1.3細胞培養 35
4.2平面壓力測試實驗流程 35
4.2.1膠原蛋白塗布 35
4.2.2進行壓力測試 35
4.3混合細胞培養測試 37
4.3.1不同環境下細胞培養 37
4.3.2細胞計數與生長曲線 37
4.4 三維多層細胞管測試 38
4.4.1前置藥品製備 38
4.4.2三維單層細胞管改良 38
4.4.3三維多層細胞管培養 40
4.4.4三維細胞管冷凍保存 41
4.5實驗儀器 41
4.5.1平面壓力測試儀器與藥品 41
4.5.2混合細胞培養測試儀器與藥品 43
4.5.3三維多層細胞管儀器與藥品 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 48
5.2混合細胞培養測試結果 48
5.2.1各類參數影響比較 49
5.3三維細胞管培養結果 50
5.3.1三維單層細胞管改良 50
5.3.2三維多層細胞管培養 52
5.3.3三維細胞管冷凍保存 54
第六章 結論 56
6.1平面壓力實驗測試 56
6.2混合細胞培養測試 57
6.2.1各類參數的影響 57
6.3三維細胞管製作 58
6.3.1三維單層細胞管改良 58
6.3.2三維多層細胞管培養 58
6.3.3三維細胞管冷凍保存 59
第七章 未來工作 60
第八章 參考文獻 61

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