2014-08-012024-05-17https://scholars.lib.ntu.edu.tw/handle/123456789/689127摘要：腦中風在已開發國家是很嚴重的醫療與經濟問題，根據WHO歷年調查顯示，中風 一直高居十大死因前三位。由於中風的致病原因十分的複雜，截至目前為止，在這領域 中雖有許多研究，但仍然無法窺見全貌。在所有中風患者中，大約有80%為缺血性中風， 時至今日，主要只有一個有效治療藥物-血栓溶解劑(tPA)可以治療缺血性中風，但是使 用血栓溶解劑會受到時間上的限制，根據最新臨床上的使用經驗，若將血栓溶解劑使用 於發病超過四個半小時的患者，除了會大大降低其治療效果，還會增加病人發生腦出血 的機率，甚至造成血腦障壁的損傷，導致神經損傷更加嚴重，因此研究其他的治療標的 來研發藥物以治療中風是十分重要的。我們之前以人類細胞激素檢測盤(human cytokine array)來測定使用血栓溶解劑前後 中風病患的血清中細胞激素濃度變化。我們發現RANTES與MIF這兩種細胞激素，在 缺血性中風發生時，濃度上升，而在使用血栓溶解劑後也明顯地降低其濃度。因此我們 認為這兩種細胞激素應該與中風患者缺血產生的發炎變化有關，因此我們為了深入研究 此二因子，會再以ELISA的方式，進行更多臨床檢體的檢測。而在基礎研究上，我們 已經初步發現此二細胞激素之拮抗劑，皆能有效改善大鼠中風後，神經損傷的面積；甚 至合併使用兩者時，似乎能有更佳的保護效果。我們會以各種動物或離體實驗來釐清這 兩種細胞激素在腦中風發生時，所扮演的角色與作用機轉，並深入研究MIF以及 RANTES在神經保護的角色。第一年目標進一步研究RANTES以及MIF拮抗劑在中風發生時神經保護之效果，並期望能找 到兩者最佳使用時機與濃度，及評估合併使用之效果。此外也會探討MIF以及RANTES 改變情形與中風病理之間的關係。(1)我們將會測量更多臨床上使用tPA癒後良好及療效欠佳的病患，其血清中RANTES 與MIF的濃度。(2)深入研究RANTES與MIF兩者在老鼠中風時產生的改變，以及給予此二細胞激素對 中風時，是否會增加神經損傷以及增加血腦障壁通透性的作用。(3)我們會用市面上可取得的此二細胞激素拮抗劑(RANTES:Met-RANTES; MIF: ISO-1)，來找出拮抗劑最佳的使用時間點與濃度。更會進一步評估，兩者合併使用時， 減少神經損傷以及降低血腦障壁通透性的作用。(4)探討短時間(24小時)以及長期(一個月）大鼠腦中風模式下，此二細胞激素拮抗劑神經 保護之作用。同時並進行動物行為的評估。(5)在大鼠腦中風模式下，測量不同時間點的腦脊髓液與血液中RANTES與MIF的變化。(6)評估兩細胞激素拮抗劑在自發性高血壓大鼠(SHR)，是否仍然具有神經保護之作用。 第二年目標我們會在各種離體與活體實驗上，以各種生化分析的方式，來找出RANTES以及 MIF在中風發生時所扮演的角色，並且與合作實驗室研究發展對此兩種細胞激素新的 拮抗劑，期望能發展出有效神經保護劑。(7)研究RANTES與MIF兩者拮抗劑，對中風神經保護的作用機轉與訊息傳遞。(8)我們會以活體或離體實驗，來篩選與合作實驗室發展新的RANTES與MIF拮抗劑， 期望能找出效果更佳的中風治療藥物。在中風治療上，雖然各大藥廠皆投入大量心血研發，但市場仍然十分需要更有效之 藥物，期盼我們對RANTES與MIF兩細胞激素，從臨床到基礎的研究能提供一個未來藥 物研發之標的。<br> Abstract: Stroke is the third leading cause of death in industrialized nations. Until now the only drug on market for stroke therapy is tissue plasminogen activator (tPA). For patients with acute stroke, intravenous tPA of stroke onset are interventions of proven benefit. A key determinant for patient selection for thrombolytic therapy is the time from stroke onset, and the efficacy for i.v. tPA is established only up to 3 hours after symptom onset. Since the plasmin produced by tPA is a non-specific protease, which not only dissolves clots but also contributes to vascular degradation and opening of the blood-brain barrier by matrix metallo proteinases, and can thereby worsen damage from reperfusion injury if given in delayed reperfusion. During ischemic stroke and subsequent reperfusion, the tight junctions of the blood-brain barrier are disrupted, resulting in the increase of vascular derived substances into the brain. Therefore, it is urged to find new targets for developing new neuroprotectants to treat stroke patients.We have preliminarily used The Human Cytokine Array to detect the serum levels in control and stroke patient (treated with tPA). The relative expression levels of 36 human cytokines were determined. Only RANTES (CCL5) and macrophage migration inhibitory factor (MIF) were up-regulated before tPA treatment and declined after tPA treatment in stroke patients. Thus, we will focus on these two chemokines and examine the roles of two chemokines in the pathology of stroke. Our preliminary result showed that RANTES and MIF antagonist treatment significantly reduced the infarct volume after stroke in rat, and combination of two antagonists had synergistic effects.Year-OneThe goal of first year is to evaluate the pathological roles ofRANTES and MIF in stroke and the neuroprotective effect ofRANTES and MIF antagonists.⑴ The serum levels of RANTES and MIF will be measured using ELISA kit in more stroke patients. (tPA responsive and tPA unresponsive patients)(2)We will examine the roles of two chemokines, RANTES and MIF, in the pathology of stroke in rats. The effects of recombinant RANTES and MIF protein on infarction volume and BBB permeability will be examined.(3)We will use the marketing compounds, Met-RANTES and ISO-1, to inhibit the action of RANTES or MIF, respectively. The best dosage and timing for these two antagonists to treat stroke will be explored. Moreover, we will combine RANTES antagonist with MIF antagonist to see the additive or synergistic effects for the treatment of stroke. The effects of antagonists on infarction volume and BBB permeability will be investigated.(4)The acute (24 h) and chronic (1 month) effects of RANTES and MIF antagonists will be examined. Several behavior tests will be performed.(5)The CSF and blood levels of RANTES and MIF will be measured at different time points following MCAO in rats.(6)The effects ofRANTES and MIF antagonists on the stroke in Spontaneously Hypertensive Rat (SHR) will be examined.Year-TwoWe will use biochemical and molecular approaches to find the signaling pathway of RANTES and MIF. This notion will be evaluated by in vitro and in vivo experiments. We will do virtual screening to find the suitable RANTES or MIF antagonist by computer modeling.(7)To explore the action mechanism of RANTES and MIF antagonist for the treatment of stroke.(8)We will collaborate with chemists to screen new RANTES and MIF inhibitor for neuroprotection both in vitro and in vivo.This project will translate clinical findings in stroke patients to basic identification of possible drug targets for the treatment of stroke.