腎臟足細胞（Podocyte）是位於腎小球毛細血管基底膜外層特殊分化的細胞，
其足突間的裂孔膜（Split Membrane）是腎小球過濾的最後一道屏障，當足細胞受損時，
足細胞的蛋白濾過屏障完整性會被破壞，造成大量蛋白尿的產生[1,2]。
本實驗將足細胞培養在孔洞直徑大小為0.02 μm氧化鋁滲透膜上(AAO membranes)，置於微流體裝置中，施加不同壓力差以模擬體內腎足細胞在血液壓力變化下過濾血液的狀態。
實驗結果，在人體體內收縮壓大於180 mmHg以上，也就是在腎小球微血管所承受壓力大於60 mmHg的時候，除了腎足細胞的足突長度明顯下降14.24 %，Synaptopodin的表現量也逐漸降低，並藉由不同大小帶有螢光蛋白質的實驗顯示，在大於60 mmHg的壓力差之下，本來不應該被濾過的500 Kd大小蛋白質被過濾出來，表示足細胞在高於人體臨界壓力之下，細胞過濾能力受損。
體內腎足細胞一旦遭受損壞，是沒有辦法再生的，所以患終端末期腎臟病患者，通常需要長期洗腎來維持體內代謝平衡，使用不同大小帶有螢光蛋白質檢測裝置修復能力實驗結果，發現受損足細胞施以正常壓力六小時後，會有13 %的修復能力。
Keywords: podocyte 、Split Membrane 、shear force、cytoskeleton、kidney failure

Chronic kidney disease (CKD) is progressively loss of renal filtration function. Here we report an in vitro model of podocyte culture that reconstitutes glomerulus filtration function and pathological behavior in response to hypertension, which is one of the leading causes of CKD. Podocytes, the glomerular epithelial cells that processes highly branched architecture essential for glomerular filtration, has been the central focus for studying CKD. Mimicking the glomerular basement membrane, podocytes grew on collagen coated anodic aluminum oxide (AAO) membranes exhibited high degree of cell spreading and fastest growth rate. To study the influence of hypertension, confluent podocytes on porous membrane was sandwiched by upper and lower chambers supplying with transmembrane pressure ΔP. The filtration function was validated using dextran. The result shows dextran in 20 kDa and 70 kDa can penetrate the podocyte membrane whereas dextran in 500 kDa was blocked until ΔP > 60 mmHg, which resembles the level of hypertension. Additionally, with the increase of ΔP, we found that more foot processes grew and reached a maximum at ΔP = 30 mmHg (19.718%) but it decays when ΔP is further increased. Moreover, analysis of mRNA expression shows synaptopodin is also down-regulated when ΔP > 40 mmHg. These results suggest that the dysfunction of renal filtration is correlated with the reduction of foot processes and synaptopodin expression but not just cytoskeletal reorganization. Taking together, our in vitro platform enables the reconstitution of renal function and investigation of CKD mechanism, with implications for drug development in the future.

目錄
摘要……………………………………………………….........................……….……...… i
致謝 ………………………………………………………………………….………....… iv
目錄…………………………………………………………………………….………..… v
圖目錄…………………………………………………………………………..………… vii
表目錄…………………………………………………………………………..………… xii
第一章 序論 …………………………………………………………………….……….…1
1.1 前言 …………………………………………………………….……………1
1.2 研究動機 ……………………………………………………….……………5
第二章 文獻探討 ……………………………………………………………………….….7
2.1 影響腎足細胞過濾能力因素………………………………………………...7
2.2 腎足細胞標記蛋...……………....................................................................10
2.3施加機械力所造成的改變…………………………………….…………….13
2.3.1 裝置設計……………………………………………..……………..13
2.3.2 滲透膜的選擇……………………………………….…………….15
2.3.3 研究目標…………………………………………….…………….16
2.4 腎足細胞與腎臟疾病關係………………………….……..………18
2.4.1發病機理……………………………………...……………………..18
第三章 實驗設計與流程…………………………………………………….……………20
3.1 腎足細胞生長速度……………………………………………...…………22
3.1.1調配培養液……………………………...…………………………22
3.1.2實驗步驟…………………………………...………………………23
3.2生長密度………………………………………… …………………………….23
3.2.1實驗步驟……………………………………...……………………23
3.3將細胞養在不同種膜類，孔洞大小和開孔率對細胞生長的影響…..…..........23
3.3.1未塗布I型膠原…………………………………………………… 24
3.3.2塗布I型膠原……………………………………………….………26
3.4模擬體環境細胞加壓實………………………………………………..………27
3.4.1 裝置設計…………………………………………………….…….27
3.4.2 加壓壓力…………………………………………….……….……28
3.4.3 細胞追蹤…………………………………….……………….……29
3.5模擬體內環境細胞加壓實驗………………………………………..…………30
3.5.1足突身長量與壓力差關係…………………………….…..………30
3.5.2蛋白質表現量遇壓力關係………………………………...………34
3.5.3使用不同大小戴有螢光蛋白質檢測裝置過濾能力………...……36
3.5.4使用不同大小帶有螢光蛋白質檢測細胞修復能力………...……39
第四章 實驗藥品與設備儀器……………………………………………………..……40
4.1實驗藥品…………………………………………………………..........……40
4.2實驗儀器與器材………………………………………………......…………41
4.3實驗儀器原理介紹……………………………………………..……………45
第五章 結果與討論……………………………………………………………….….......49
5.1生長速度曲線……………………………………………………….……...49
5.2細胞密度與生長曲線關係………………………………………................51
5.3將細胞養在不同種類滲透膜、孔洞大小和不同開孔率對細胞生長的影響……………….………...........................................................................…52
5.3.1 未塗布I型膠原……………………………………………...........52
5.3.2 塗布I型膠原………………………………………………….......56
5.4模擬體環境細胞加壓實驗………………………………………………...60 5.4.1 足突伸長量與壓力差關係………………………………..........60
5.4.2蛋白質表現量與壓力關係…………………………………….…63
5.4.2.1 Synaptopodin…………...................................................63
5.4.2.2 Actin-4……………………………………………..……63
5.4.3使用不同大小戴有螢光蛋白質檢測裝置過濾能力…………...…64
5.4.4使用不同大小帶有螢光蛋白質檢測細胞修復能力…….............. 67
第六章 結論……………………………………….………………………………...…...…69 第七章 未來工作……………………………………………………………….………..…70 第八章 參考文獻……………………………………………………………………...……74

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