模擬人體血管之剪應力-拉伸裝置用於研究內皮細胞的行為

Abstract

本研究之目的為開發一個裝置來模擬人體動脈血管的微流體系統，因為流體剪應力(FSS, fluid shear stress)和循環拉伸(CS, cyclic stretch)是心血管系統中主要的機械刺激，因此欲整合FSS和CS在此微流體裝置上。將待觀察之血管內皮細胞(ECs, endothelial cells)注射至微流體裝置中並將其放入二氧化碳培養箱(Incubator)內，再藉由所建立的可程式化氣壓源系統、注射幫浦(syringe pump)和活細胞培養系統結合顯微鏡，可以即時(real-time)觀察ECs在受到不同流場(連續流、往復流和脈衝流)的形態與黏附的變化。 本研究是使用黃光微影製程和軟微影技術(Soft Lithography)來製作微流體裝置，並選用聚二甲基矽氧烷(PDMS, Polydimethylsiloxane)作為材料，因為PDMS具有光學透明、通透性、彈性佳和生物相容性佳的特性。將PDMS和玻璃使用氧電漿機(Oxygen Plasma)進行表面改質後結合成封閉的微流道，即完成實驗使用之微流體裝置。PDMS是用於微流體裝置設計的有用材料，提供了方便、有效和低成本的製程方法，對於器官晶片等實驗室晶片的領域可以快速的發展。 本研究使用牛動脈內皮細胞(BAECs, bovine aortic endothelial cells)進行實驗，模擬內皮細胞受到血流而產生流體剪應力，以及血壓造成的循環拉伸應變，並觀察內皮細胞受到此機械刺激後其形態和功能的改變，主要包括內皮細胞會調整細胞骨架(cytoskeleton)的方向，以及內皮細胞會出現樁蛋白(paxillin)分佈於細胞的周圍與應力纖維(stress fibers)末端，因此研究流體剪應力和循環拉伸應變如何影響內皮細胞有助於我們了解改變機械環境時，內皮細胞的形態、功能改變和相關心血管疾病的發展，未來可以開發藥物治療之方法來解決心血管疾病的問題。

The objective of this study was to develop a device to mimic a microfluidic system of human arterial blood vessels. This device integrates fluid shear stress (FSS) and cyclic stretch (CS) which are the two major mechanical stimulations in cardiovascular systems. The endothelial cells (ECs) were injected into the microchannel and placed into an incubator (37 °C, 5 % CO2), using a programmable air pressure, a syringe pump and a stage top incubator(STR) combined with the microscope. The system can realize real-time observation of dynamic morphological change of cells in different flow fields (continuous flow, reciprocating flow and pulsatile flow). In our study, microfluidic device was made by polydimethylsiloxane (PDMS) using soft lithography process, PDMS is an ideal material for this device since it is optically transparent, elastic and biocompatible. We expose the PDMS and glass to an air plasma, surface-oxidized PDMS can seal to glass to enclose the channel, which is completed our microfluidic device. PDMS use as materials reduces the time, complexity, and cost of prototyping and manufacturing, and the field of Lab on a Chip can be rapidly developed. This study used bovine aortic endothelial cells (BAECs) are subjected to haemodynamic forces in the form of fluid shear stress and cyclic stretch resulting from blood flow and blood pressure. The EC change in function in response to mechanical stimuli, which is also accompanied by an apparent morphological change, including ECs align their cytoskeleton proteins with the blood flow direction and paxillin redistribution to the cell periphery. Investigations on how ECs respond to combined shear stress and cyclic stretch will help us to better understand how altered mechanical environment affects ECs function, morphological and associated cardiovascular disease development. Understanding the relationship between mechanical stimuli and vascular pathology can lead to improved treatments.