具奈米孔洞仿生膜之微流體系統應用於研究壓力對腎臟足細胞生理以及病理上的影響

A Micro Fluidic System Integrated With Biomimetics Nano-Membrane for Studying Podocyte Response to Different Pressures

呂芷萱

指導教授 ： 曾繁根

國立清華大學/工學院/奈米工程與微系統研究所/碩士(2014年)

摘要

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

關鍵字

足細胞 ； 裂隙 ； 細胞骨架

並列摘要

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.

並列關鍵字

podocyte ； Split Membrane ； cytoskeleton

參考文獻

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