- 學位論文
內皮細胞施以剪切應力於不同幾何形狀晶片模擬缺血性腦中風模型
Applying Shear Stress to Endothelial Cells on Microfluidic Chips with Various Geometries to Simulate Ischemic Stroke Models
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摘要
近年社會變遷，人們生活壓力、型態、飲食均增加且改變，直接提高中風、高血壓、心肌梗塞等疾病風險。中風源於血栓形成，導致血管栓塞，引起腦部缺血及死亡症狀，因此預防和消除血栓的研究至為重要。目前，血栓藥物篩檢和評估仍使用動物實驗方法，傷害動物且由於數據再現性及複雜的生理交互作用，導致研究結果難與臨床數據整合。因此，發展替代動物實驗方法對於血栓研究極為重要。 本研究的宗旨是於體外模擬不同彎曲角度的血管晶片，並在其彎曲處觀察血栓形成的差異。研究方法整合了微流體系統和內皮細胞模型培養技術，利用微流道覆蓋內皮細胞來模擬血管內環境，並觀察晶片於不同角度之彎曲處血栓的形成和溶栓過程。除此之外，本實驗亦利用ROS螢光染劑搭配注射幫浦將培養液 (M199)注入內皮細胞覆蓋之血管晶片中，即時檢測血管晶片中內皮細胞受到剪切力之影響。連續的剪切力將會造成晶片內局部內皮細胞受到損傷，再利用注射幫浦將新鮮血液注入細胞損傷的血管晶片中，同時對血小板(platelet)與纖維蛋白(Fibrin)進行染色，以觀察血栓形成，並比較不同彎曲角度晶片血栓的形成變化與給予藥物後血栓的消融情形。 在本研究中，我們選擇使用雷射雕刻機在薄膜上製作微流道模具，隨後利用(Polydimethylsiloxane, PDMS)進行翻模。PDMS具有優越的光學透明性、通透性、彈性以及生物相容性等特性，因此成為微流道翻模的主要材料。接下來，透過氧電漿機對PDMS和玻璃進行改質處理，再將其接合固定，完成封閉的微流道晶片。這個製程具有低成本、方便且高效的優勢，有效提升了微流道晶片的製作效率和製程良率。 總結研究成果，我們成功建立了一個可控制的系統，能夠在欲觀察的位置產生血栓。透過微流道的不同彎曲角度，我們在晶片上實現了多種不同程度的血栓形成。未來，我們期望這種方法可以替代或補充動物實驗，提供一個更可靠、更準確的血栓模型。這有望為血栓研究提供更進一步的洞見，促使相關領域的發展。
並列摘要
In recent years, societal changes have led to increased life stress, altered lifestyles, and dietary modifications, directly raising the risks of diseases such as stroke, hypertension, and myocardial infarction. Stroke, resulting from thrombus formation causing vascular occlusion, leads to symptoms of cerebral ischemia and death. Therefore, research on the prevention and elimination of thrombosis becomes crucial. Currently, screening and evaluation of thrombolytic drugs still rely on animal experiments, causing harm to animals, and the reproducibility of data along with complex physiological interactions make it challenging to integrate research results with clinical data. Hence, the development of alternative methods to animal experiments is of utmost importance for thrombosis research. The aim of this study is to establish a controllable system for generating blood clots at specific locations. The research methodology involves integrating microfluidic systems and endothelial cell culture techniques to simulate the vascular environment, observing clot formation and dissolution in the curved regions of the chip. Using an injection pump to introduce culture medium (M199) into endothelial cell-seeded chips, the impact of shear forces on endothelial cells is monitored over time using a ROS dye. By introducing blood into the damaged chips and staining platelets and fibrin, clot formation is observed. The study then compares the clot formation and dissolution states on chips with different bending angles to assess the degree of occlusion. In this study, we opted to use a laser engraving machine to create microchannel molds on thin films, followed by replication using Polydimethylsiloxane (PDMS). PDMS, with excellent optical transparency, permeability, elasticity, and biocompatibility, serves as the primary material for microchannel replication. Subsequently, PDMS and glass are modified using oxygen plasma treatment, and the two are bonded and fixed, completing the enclosed microfluidic chip. This process offers advantages such as low cost, convenience, and efficiency, effectively improving the production efficiency and process yield of microfluidic chips. In conclusion, we have successfully established a controllable system capable of generating blood clots at specific locations. Through varying the bending angles of microchannels, we achieved different degrees of clot formation on the chip. In the future, we hope that this method can serve as an alternative or complement to animal experiments, providing a more reliable and accurate thrombosis model. This is expected to contribute to gaining deeper insights into thrombosis research, fostering advancements in related fields.
參考文獻
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