| 摘要: | 缺血性中風後的再灌注會因免疫細胞的浸潤，破壞腦部原本之細胞外基質進而使血腦屏 障受損。有研究指出，缺血性中風前的運動訓練能降低金屬蛋白脢的表現，降低缺血性 中風對於腦部細胞外基質的喪失，進而降低腦部的傷害。然而，中風後的運動治療對於 腦部細胞外基質影響的研究卻付之闕如。本研究的初步結果顯示，缺氧後短時間內腦部 血管的細胞外基質會因金屬蛋白脢的表現而喪失，但長時間後腦內的細胞外基質反而會 過量表現。由於細胞外基質如第一型膠原蛋白的過量表現在其他器官與纖維化有關，因 此，本計畫預計以三年的時間探討運動治療對於此腦纖維化的影響。分年目標如下: 第一年、 觀察中風後腦部細胞外基質的變化及運動治療:首先，透過阻斷middle cerebral artery 血流誘發B6 小鼠及SD 大鼠腦部缺血性中風，於120 分鐘後放開恢復血 流，引發再灌注的傷害。小鼠及大鼠分組為每天游泳30 分鐘及對照組，於手術 後48 小時開始運動訓練，並於第7、14、21 及28 天後犧牲。腦組織將以免疫 螢光染色觀察細胞外基質的變化以及遷徙到腦內的免疫細胞種類。初步針對不 同細胞外基質觀察缺血再灌流對細胞外基質的影響，主要觀察細胞外基質為 collagen I、collagen IV、laminin γ1 及fibronectin，並利用細胞表面標記(cell surface marker)觀察T 細胞、巨噬細胞、中性球、星狀細胞及小神經膠質的數 量及活化情形。 第二年、 觀察腦部細胞外基質過量表現的機制: 以第一型膠原蛋白為例，前人研究指出 最有可能表現第一型膠原蛋白的細胞為周圍細胞(pericytes) 、小神經膠細胞 (microglia)或是內皮細胞。第二年的研究將以免疫螢光染色為主，利用第一型 膠原蛋白的染色搭配細胞表面標記，觀察有可能製造第一型膠原蛋白的細胞為 何。體外實驗則將分離小鼠之周圍細胞、小神經膠細胞及內皮細胞做體外培養， 觀察缺氧再灌注的壓力下何種細胞會表現第一型膠原蛋白。 第三年、 觀察運動訓練對細胞外基質過量表現影響的機制:本研究的第三年體外實驗將 根據前兩年的結果，觀察運動訓練是透過哪些途徑，改變長期中風後細胞外基 質的過量表現。初步將觀察金屬蛋白脢以及細胞激素，如與發炎相關的 TNF-α，以及與纖維化相關的TGF-β 等等。 綜合以上所述，本研究的目標是觀察運動訓練對腦組織中細胞外基質的調控，以及對 免疫細胞浸潤的影響。此方面知識的建立將有助於了解中風後的運動對於腦部的幫助。
The reperfusion after ischemic stroke in brain induces massive immune cells infiltration, sequentially damages the extracellular matrix (ECM) of blood brain barrier (BBB). Previous studies indicated that pre-conditioning exercise can reduce metalloprotease (MMP) expression and then protect BBB damage induced by stroke. However, there is no study evaluates the effect of post-stroke exercise on changes of ECMs. In our preliminary results, ECM loss was found in acute stage because of MMP expression, but large amount of ECM, such as collagen type I, expressed in stroke area in 7 days after stroke. Because excess ECM is related to fibrosis, we wonder if exercise training can modulate ECM overexpression. Thus, the aim of this proposal is to elucidate the effect of exercise on ECM after ischemic/reperfusion injury. The goals in each year are, 1st year. Observe the changes of ECMs after exercise training in ischemic brain injury Through 120 min middle cerebral artery occlusion (MCAo), the ischemic/reperfusion injury model in brain will be established. The mice and rats will be separated into two group, one with 30 min swimming exercise everyday start at 48h postop and the control group without exercise. The brain will be taken at 7, 14, 21, and 28 days after exercise started. The changes of infarct size, ECMs including collagen I, collagen IV, laminin γ1 and fibronectin, will be observed through immunofluorescence staining. In addition, the infiltration and activation of T cells, macrophages, neutrophils, astrocytes an microglia will be also investigated. 2nd year. Investigate the mechanism of ECMs overexpression after ischemic/reperfusion injury Take collagen I as example, previous studies indicate the cells which might produce collagen I in brain are pericytes, microglia or endothelial cells. The works in second year will be performed with double staining of cell surface marker and ECM. The cells which producing excess ECMs after ischemia will also be confirmed by ex vivo experiments. 3rd year. Characterize the mechanism of exercise on ECM changes after stroke Base on the results of first two years, the study of third year will focus on how exercise changes the ECM expression of induced by stroke. We will observe the expression and activity of MMP2 and MMP9. Furthermore, cytokines relates to inflammation and fibrosis, such as TNF-α and TGF-β will be investigated. In summary, we will investigate the effect of post-stroke exercise training on infarct size, ECMs changes, and the further effects on immune cells infiltration. An understanding of this important mechanism may contribute to basic knowledge in exercise on stroke patients. |
