微流體剪應力對羊水幹細胞分化為內皮細胞之影響

Abstract

幹細胞治療在心血管疾病的應用上漸漸受到關注，利用微機電製程製造的微晶片，提供比公分尺寸的實驗更接近人體的培養環境，例如細胞和細胞間的距離以及類似人體血管的培養體積，在這理論基礎下，微機電技術製造的微晶片提供流體剪應力的刺激，可以加速羊水幹細胞分化成內皮細胞 內皮細胞有沿著流場方向排列的特性，匯整人類羊水幹細胞在12 dyne/cm2 剪應力刺激24小時後的排列方向統計資料，剪應力刺激3小時後的排列角度分布和24小時的結果接近，表示其有相近的分化結果，此外，剪應力12 dyne/cm2 作用3小時以及24小時後，血小板與內皮細胞貼附分子(platelet endothelial cell adhesion molecule ,PECAM-1) 和 溫偉伯因子(von Willebrand Factor, vWF)都有明顯的表現，經過3小時的剪應力刺激，血小板與內皮細胞貼附分子, 溫偉伯因子以及血管內皮生長因子受體(vascular endothelial growth factor-2 receptor, VEGFR)的基因表現量都超過10倍以上，另外經過剪應力刺激後，在基質膠(matrigel) 基底上，觀察到低密度脂蛋白的堆積以及網狀結構形成，細胞在剪應力12 dyne/cm2 刺激下，分化程度比3, 6, and 18 dyne/cm2來的高 在巨觀尺寸的實驗架構下，需要數天才能達到分化的結果，而微流道系統成功的提供了快速分化的環境，人類羊水細胞在微流道系統的培養環境下，3小時就有明顯分化結果，本研究顯現出其在幹細胞的治療以及心血管疾病的研究上的潛力

Stem cell therapies for cardiovascular diseases are of great growing interest. Micro-electro-mechanism-system (MEMS) fabricated chips provide more in vivo like cell-to-cell distance and culturing volume than that in centimetre-scaled experiments. In this thesis, we are able to accelerate the process of endothelial differentiation of human amniotic fluid stem cells (hAFSCs) by the stimulation of fluidic shear stress in a MEMS-fabricated micro device. Cells aligning to flow directions is a known property of endothelial cells. hAFSCs affected by 12 dyne/cm2 of shear stress were compiled statistics for the arrangement angles toward the flow direction in a 24-hour period. The angular distribution of cells after 3 hours of stress stimulation approaches to that of cells after 24 hours, indicating similar degrees of endothelial differentiation. Both cell samples stimulated by 12 dyne/cm2 stress after 3 hours and 24 hours exhibit protein expressions of platelet endothelial cell adhesion molecule (PECAM-1) and von Willebrand Factor (vWF). Over ten folds of gene expressions in PECAM-1, vWF and vascular endothelial growth factor-2 receptor (VEGFR) appeared after 3 hours stimulation. Uptakes of ac-LDL and formation of tube-like structure on Matrigel were also observed after shear stress stimulation. Cells stimulated by 12dyne/cm2 shear stress have higher degrees of differentiation than those stimulated by 3, 6, and 18 dyne/cm2 shear stress. Utilizing 3 hours to achieve differentiation instead of days in macro-scale experiments, this micro fluidic system has been proved successful for a rapid shear stress stimulation of hAFSCs differentiation into endothelial cells. This research shows its great potential in stem cell therapy and cardiovascular disease studies.