足細胞為人體腎臟內一種特殊上皮細胞，對於維持腎小球過濾系統扮演重要角色。當足細胞受到損傷並丟失，會導致許多腎臟疾病的發生。藥物療法為ㄧ般臨床上用於治療足細胞疾病，雖然能減緩腎功能下降的問題，但仍難以恢復腎功能並阻止慢性腎臟病的發展。而細胞療法為一具有潛力的替代療法，先前許多研究使用細胞療法作為足細胞造成腎損傷的治療方法，雖然於結構修復上看到成效，卻發現無法於腎功能上看到顯著恢復，仍存在諸多限制。本篇論文希望透過外源性足細胞的移植，來補充丟失的足細胞的，為了達更有效的移植，我們將足細胞與內皮細胞共培養模擬腎絲球微環境，並加入間葉幹細胞輔助製成三維複合細胞球體之系統。在體外實驗中，我們製作了四種不同種類細胞球相互比較，探討加入間葉幹細胞與內皮細胞對於足細胞之影響，由基因及蛋白結果分析，間葉幹細胞與內皮細胞各自對於足細胞扮演促存活及助分化重要之角色，並且透過免疫螢光染色證實我們三維複合細胞球體具有分泌許多生長因子及細胞外基質之潛能，因此我們選用三種細胞共培養細胞球，作為後續體內實驗觀察之重要組別；於體內實驗結果，我們透過組織免疫螢光染色追蹤足細胞，證實我們的外源性足細胞至少能在腎內存活長達至兩周，但卻無明顯於腎絲球處觀察到外源性足細胞的存在；而在高血壓腎病模式小鼠中觀察並評估兩周內腎功能變化情形，發現打入細胞球既沒有明顯腎功能衰竭的變化，還顯著降低蛋白尿的情形。
由結果總結，我們可以製造出大量且均一性高的三維細胞球，並且對於改善腎功能具有極大的潛力。

Podocytes are highly differentiated epithelial cells that play an important role in maintaining glomerular filtration barrier in human kidney. Podocyte injury followed by depletion is the major cause for the initiation and pathological progression of multiple types of kidney diseases. The mainstream pharmaceutical-based approaches often fail to effectively delay or reverse the progression of nephropathy. Kidney transplantation and dialysis are the ultimate life-saving options for patients who develop renal failure. However, kidney transplantation is severely hindered by the shortage of donors, while dialysis significantly diminishes the patients’ quality of life. Cell therapy has been considered promising for treating podocyte injury. Nevertheless, the resultant therapeutic efficacy in terms of renal function improvement is limited, possibly owing to significant loss of engrafted cells. Herein, three-dimensional (3D) hybrid cell spheroids composed of podocytes, mesenchymal stem cells and vascular endothelial cells were designed to replenish the podocyte population by cell transplantation. By creating a glomeruli-like microenvironment, the expression of multiple genes of basement membrane and growth factors that are highly associated to podocyte maturation was significantly enhanced. Our in vivo results demonstrated that podocytes that were intrarenally transplanted in the configuration of 3D hybrid cell spheroids exhibited improved engraftment efficiency. Moreover, the proteinuria of transgenic mice with chronic renal disease was effectively reduced by treating with the developed 3D hybrid cell spheroids, thus demonstrating their potential for repairing the injured kidneys.

目錄
摘要 i
Abstract ii
致謝 iii
目錄 iv
圖目錄 vii
表目錄 x
第一章、 緒論 - 1 -
1.1 腎小球構造及功能 - 1 -
1.1.1 足細胞(podocyte) - 2 -
1.1.2 腎內皮細胞(glomerular endothelial cell, GEC) - 4 -
1.1.3 腎小球內系膜細胞(mesangial cell, MC) - 5 -
1.1.4 腎小球過濾屏障(glomerulus filtration barrier, GFB) - 5 -
1.2 誘發足細胞丟失的成因 - 7 -
1.2.1 藥物劑量的累積(drug accumulation) - 7 -
1.2.2 物理應力刺激(mechanical stress) - 7 -
1.3 臨床療法 - 8 -
1.3.1 血液透析 - 8 -
1.3.2 藥物控制 - 9 -
1.4 腎功能指標 - 9 -
1.4.1 血清肌酸酐(creatinine) - 9 -
1.4.2 蛋白尿(proteinuria) - 9 -
1.5 細胞療法 - 10 -
1.5.1 間葉幹細胞移植後的旁分泌作用促進腎修復 - 10 -
1.5.2 誘導幹細胞分化為腎細胞作為補充使腎組織再生 - 10 -
1.6 永生化足細胞(immortalized podocyte) - 12 -
1.7 間葉幹細胞 - 12 -
1.8 三維複合式細胞球體 - 14 -
1.8.1 懸掛式培養(hanging drop) - 14 -
1.8.2 微圖案陣列熱敏性水凝膠 - 15 -
1.8.3 甲基纖維素 (methylcellulose, MC) - 17 -
1.9 研究動機與實驗目的 - 19 -
第二章、 材料與方法 - 22 -
2.1 細胞培養 - 22 -
2.2 甲基纖維素製備 - 23 -
2.3 三維複合細胞球製備 - 24 -
2.4 細胞球遷徙試驗(Cell migration assay) - 25 -
2.5 細胞球存活/死亡(Live/Dead) - 26 -
2.6 細胞免疫螢光染色 - 26 -
2.7 細胞球免疫螢光染色 - 27 -
2.8 即時聚合酶連鎖反應(Real-time PCR) - 28 -
2.9 西方墨點法(Western blot) - 34 -
2.10 動物模型的建立 - 38 -
2.11 組織處理 - 38 -
2.12 組織免疫螢光染色 - 39 -
2.13 蘇木精伊紅染色(haematoxylin & eosin, H & E) - 40 -
2.14 腎功能檢測 - 40 -
2.15 統計分析 - 41 -
第三章、 實驗結果與討論 - 42 -
3.1 細胞球最佳培養條件之分析 - 42 -
3.2 細胞球形態鑑定 - 44 -
3.3 比較不同種類細胞球分泌促生長因子的能力 - 46 -
3.4 比較不同種類細胞球間足細胞分化基因表現的能力 - 49 -
3.5 比較不同種類細胞球間分泌細胞外基質的能力 - 50 -
3.6 不同細胞球內足細胞的遷徙能力 - 52 -
3.7 三維複合細胞球之特性鑑定 - 53 -
3.8 細胞球過針筒之細胞存活分析 - 54 -
3.9細胞球的遞送效用(Cell retention) - 55 -
3.10 細胞的移植於長時間觀察(Cell engraftment) - 57 -
3.11 注射對於腎組織結構的影響 - 58 -
3.12 腎功能影響評估 - 60 -
第四章、 結論 - 62 -
參考文獻 - 63 -

圖目錄
圖 1-1 腎臟中腎元的構造[1]。 - 1 -
圖 1-2 腎小球的構造[2]。 - 2 -
圖 1-3 足細胞的結構[5]。 - 3 -
圖 1-4 腎小球過濾系膜之構造[6]。 - 3 -
圖 1-5 腎內皮SEM孔洞狀結構圖[8]。 - 4 -
圖 1-6 血管內皮上醣蛋白之結構[10]。 - 4 -
圖 1-7 腎小球過濾屏障[2]。 - 6 -
圖 1-8 腎小球過濾屏障損傷引起的蛋白尿[9]。 - 6 -
圖 1-9異常血壓造成足細胞損傷與足突消失及腎小球硬化的關聯示意圖[22]。 - 8 -
圖 1-10 腎前驅細胞移植用於腎再生的未來發展方向[36]。 - 11 -
圖 1-11 間葉幹細胞之特性與功能[49]。 - 13 -
圖 1-12 懸掛式培養的細胞球成球過程[57]。 - 15 -
圖 1-13 懸掛式系統所產生之podocyte與MSC共培養細胞球[54]。 - 15 -
圖 1-14 微圖案陣列水凝膠置備過程。 - 16 -
圖 1-15 利用溫度敏感性水凝膠產生自組裝之三維幹細胞球體[58]。 - 16 -
圖 1-16 甲基纖維素成膠過程及其分子結構示意圖[62]。 - 17 -
圖 1-17 甲基纖維素在乾燥與水相接觸下的物理結構[65]。 - 18 -
圖 1-18 三維複合細胞球體製備示意圖。 - 18 -
圖 1-19 實驗設計架構圖。 - 21 -
圖 2-1 細胞球製備流程圖。 - 25 -
圖 2-2 TaqMan probe在即時聚合酶連鎖反應中的螢光偵測原理。 - 29 -
圖 2-3 Reverse transcriptase PCR thermal profile (hr：mm：ss)。 - 31 -
圖 2-4 Real-time PCR thermal-cycling profile (hr：mm：ss)。 - 33 -
圖 2-5 三明治夾法。 - 35 -
圖 3-1 三維細胞球體在水膠上培養一天之相位圖結果。 - 42 -
圖 3-2 三維細胞球體在水膠上培養天數觀察之Live/Dead染色結果。 - 44 -
圖 3-3 三種細胞的型態鑑定。 - 45 -
圖 3-4細胞球大小。(a.) 螢光定性圖 (b.) Image J定量圖 - 45 -
圖 3-5 不同種類細胞球於共軛膠顯微鏡下細胞內不同層細胞分佈之結果。 - 46 -
圖 3-6 利用qPCR分析不同種細胞球間生長因子Vegfa之基因相對定量表現。 - 47 -
圖 3-7 利用qPCR分析不同種細胞球間生長因子VEGFA之基因相對定量表現。 - 48 -
圖 3-8 利用qPCR分析不同種細胞球間生長因子HGF之基因相對定量表現。 - 49 -
圖 3-9 利用qPCR分析不同種細胞球間足細胞分化因子之相對定量表現。 - 50 -
圖 3-10 細胞球分泌生長因子能力。(a.) western blot蛋白定性結果圖 (b.) Laminin定量圖 (c.) fibronectin定量圖 - 51 -
圖 3-11 細胞球的足細胞遷徙能力。 (a) 螢光定性圖 (b.) Image J定量分析足細胞於細胞球中心向外移動距離 - 52 -
圖 3-12 PME spheroid生長因子及驅化因子螢光染色表現圖。(a.) 促存活因子 (b.) 細胞基質衍生因子……………………………………………….. - 53 -
圖 3-13 PME spheroid螢光染色之ECM特性鑑定。(a.) 上皮細胞黏附因子 (b.) 基底膜基質 (c.) 間質基質 - 54 -
圖 3-14 細胞球通過針頭之Live/Dead染色結果。 - 54 -
圖 3-15 PME spheroid留存於腎組織冷凍切片之免疫螢光染色圖。 - 56 -
圖 3-16 PME spheroid留存於腎組織冷凍切片之其他切面疫螢光染色圖。 ..- 57 -
圖 3-17 注射後兩週腎組織冷凍切片之螢光染色結果圖。(a.) WT1在腎皮質層中螢光染色圖 (b.) SV T antigen在腎皮質層中螢光染色圖 - 58 -
圖 3-18注射細胞對於腎組織結構影響之H & E染色圖。 - 59 -
圖 3-19 腎功能測定。(a.) 血清肌酸酐 (b.) 蛋白尿 - 61 -
表目錄
表 2-1 細胞球免疫螢光染色之一級抗體列表。 - 28 -
表 2-2 細胞球免疫螢光染色之二級抗體列表。 - 28 -
表 2-3 RT-PCR試劑配置配方。 - 31 -
表 2-4 TaqMan probe。 - 32 -
表 2-5 real-time PCR試劑配置配方。 - 32 -
表 2-6 Western blot一級抗體列表。 - 36 -
表 2-7 Western blot二級抗體列表。 - 36 -
表 2-8 Western blot 所需之溶液配置配方。 - 36 -
表 2-9 組織螢光染色一級抗體列表。 - 40 -

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