- 學位論文
建立動物和細胞實驗模式以評估血腦障壁功能之研究
ESTABLISHMENT OF ANIMAL AND CELL MODELS TO EVALUATE THE FUNCTIONS OF THE BLOOD-BRAIN BARRIER
摘要
血腦障壁存在週邊循環系統與中樞神經系統間，它是由一連串的大腦血管內皮細胞藉由 tight junctions所組成，負責調節離子平衡、養分運輸和阻擋有害物質進入腦中。疾病進展的過程，慢性疾病例如阿茲海默症、多發性硬化症，急性症狀例如中風或腦創傷會造成血腦障壁失去功能，進而破壞中樞神經系統。在過去的動物實驗模式中，對於疾病進展過程症狀之一慢性發炎對於血腦障壁的影響，以及急性的腦缺血再灌流後，對於血腦障壁的通透性還未有明確的研究。因此，本篇研究將建立動物及細胞實驗模式以評估血腦障壁的功能正常與否。 動物模式方面我們使用 menadione 作為活性氧物質的提供者，以模擬長時間氧化壓力傷害造成慢性發炎，雙邊總頸動脈栓塞 (Bilateral common carotid artery occlusion; BCAO) 及右側大腦中腦動脈栓塞 (middle cerebral artery occlusion ; MCAO) 的手術方式接近人發生急性缺血性中風的情況。細胞模式是將腦血管內皮細胞單獨或與星狀細胞共同培養，觀察 monolayer 完整性及 ZO-1 形成情形。結果顯示，不論menadione、 MCAO 或 BCAO 組皆會導致血腦屏障破壞，而預先給予白藜蘆醇和維他命 C 可以改善血腦屏障破壞。利用2,3,5-triphenyltetrazolium chloride (TTC) 染色觀察腦受損區域，在 MCAO 組會看到明顯白色區域，表示此區細胞壞死。此外，menadione 會造成腦血管內皮細胞及 tight junction 破壞。從細胞培養型態來看，兩種培養模式皆會形成緻密 monolayer，又以共同培養的細胞型態看起來更為緊密排列。不論是單獨培養腦血管內皮細胞或共同培養細胞在第二天開始有 ZO-1 表現，共同培養情況下會有較多的 ZO-1表現，顯示 monolayer 更為完整。 由本研究成果顯示，在動物模式上，menadione 注射模式可作為慢性發炎去影響血腦障壁的研究模式，全腦性腦損傷BCAO 模式、區域性腦損傷MCAO 模式皆會造成血腦障壁不同程度的傷害， menadione 更會藉由破壞腦內皮細胞及tight junction protein使得血腦障壁通透度上升;在細胞模式上，腦血管內皮細胞間隨天數增加 tight junction protein的形成越趨完整。綜合實驗結果，這四種模式可作為日後探討抗氧化劑對於血腦障壁保護功效及機制的模式。
並列摘要
The blood-brain barrier (BBB) is between the central nervous system (CNS) and the peripheral circulatory system mainly consists of cerebral endothelial cells (CECs) that form compact tight junctions as a selective physical barrier. It functions as a dynamic regulator of ion balance, a facilitator of nutrient transport, and a barrier to potentially harmful molecules. Disruption of the BBB is a critical event in the development and progression of several diseases that affect the CNS such as neurodegenerative disorders, stroke and traumatic brain injury. Therefore, in this study, we attempted to establish animal and cell models to evaluate the functions of the BBB. In the animal study, we used menadione as a generator of oxidative stress. Bilateral common carotid artery occlusion (BCAO) and Right middle cerebral artery occlusion (MCAO) mimicked ischemia/reperfusion (I/R). The results suggested that menadione-treated, MCAO, BCAO groups led to BBB disruption. Pretreatment with resveratrol and vitamin C improved the BBB disruption. In addition, resveratrol enhanced numbers of zonula occludens-1 (ZO-1) compared with menadione-treated group. To provide an in vitro system for studying BBB functions, we have developed a process of culture at CECs with or without astrocytes in dishes. In order to detect tight junction formation, the cells formed monolayer and then immunofluorescent examination of ZO-1. These results showed that ZO-1 typically located in tight junctions of endothelial layers at 3nd day. In conclusion, the BBB permeability was increased by menadione, BCAO, and MCAO. Coculture of CECs and astrocyte formed a tighter monolayer than culture of CECs did. Through characterization of the models we have established that mice are suitable for studing protective effects of the antioxidant in BBB.