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系統識別號 U0007-1408201315354700
論文名稱(中文) Resveratrol和其衍生物保護glutamate誘導神經膠質 星狀細胞CTX TNA2死亡之探討
論文名稱(英文) The Protective Effects of Resveratrol and Its Derivatives on Glutamate-Induced Cell Death in CTX TNA2 Astrocyte Cells
校院名稱 臺北醫學大學
系所名稱(中) 醫學科學研究所
系所名稱(英) Graduate Institute of Medical Sciences
學年度 101
學期 2
出版年 102
研究生(中文) 高郁柔
研究生(英文) Yu-Rou Gao
學號 M120100040
學位類別 碩士
語文別 中文
口試日期 2013-06-20
論文頁數 65頁
口試委員 指導教授-施純明
委員-葉添順
委員-馮琮涵
中文關鍵字 resveratrol, piceatannol, oxyresveratrol, rhapontigenin, pteorstilbene, AMPK, 活性氧化物, GSK-3β, mitochondrial dysfuction, 細胞自噬, 細胞凋亡 
英文關鍵字 resveratrol, piceatannol, oxyresveratrol, rhapontigenin, pteorstilbene, AMPK, ROS, GSK-3β, mitochondrial dysfuction, autophagy, apoptosis 
學科別分類
中文摘要 根據2012年行政院衛生署統計,事故傷害位居十大死因的第六位,其中創傷性腦損傷(traumatic brain injury, TBI)占了事故傷害的12.5 %,並造成其中55 %的病患死亡,且臨床上創傷性腦損傷也是造成全球45歲以下病患癱瘓甚至死亡的一大因素,因此發展治療創傷性腦損傷藥物是非常重要的。當腦部受到外力傷害時,神經傳導物質glutamate會大量釋放造成興奮性毒性(excitotoxicity)、神經發炎反應(neuroinflammation)和神經細胞死亡。本實驗室已證實白藜蘆醇(resveratrol)藉由抑制活性氧化物(reactive oxygen species, ROS)/glycogen synthase kinase-3β(GSK-3β)/mitochondrial damage signaling,進而增加膠質星狀細胞CTX TNA2的存活,然而其藥物結構與藥理活性的相關性,則未有相關研究。因此本論文將探討resveratrol及其四種衍生物piceatannol、oxyresveratrol、rhapontigenin、pterostilbene對於神經細胞保護作用以及治療TBI療效的差異。本實驗利用glutamate處理CTX TNA2 細胞為TBI模式,初步結果顯示CTX TNA2細胞經由8 mM glutamate處理48小時後,resveratrol和其衍生物皆可減少glutamate的細胞毒性。利用流式細胞儀搭配acridine orange及annexin V/PI染劑分別偵測autophagy及apoptosis的比例,結果顯示resveratrol及其衍生物皆可降低autophagy以及apoptosis,達到保護神經膠質細胞的作用,其中又以rhapontigenin的效果最佳。此外,利用DCFH-DA、HEt和DHR123染劑偵測細胞內ROS變化,發現resveratrol和rhapontigenin具有抑制ROS產生的效果。且透過西方墨點法和JC-1偵測粒線體膜電位發現,resveratrol和rhapontigenin可促使Ser9-GSK-3β磷酸化,降低粒線體去極化作用,保護細胞免於glutamate導致的粒線體損傷。此外resveratrol和rhapontigenin可維持AMP-activated protein kinase (AMPK)的磷酸化;且AMPK抑制劑compound c可造成Ser9-GSK-3β的去磷酸化,並減少resveratrol和rhapontigenin的細胞保護作用。綜合上述實驗結果證明resveratrol和rhapontigenin具有神經保護作用,且過程可能藉由抑制ROS,誘導AMPK的活化,進而抑制GSK-3β的活性,降低mitochondrial dysfunction,最終減少glutamate誘導的autophagy與apoptosis。且由MTT assay顯示,rhapontigenin抑制glutamate導致的毒性效果最好,顯示若在resveratrol B環上將hydroxy group取代為methoxy並增加一hydroxyl group,可提高resveratrol的保護效果。因此,本研究期望可提供臨床上開發治療創傷性腦損傷藥物的新方向,透過有效的減少神經系統的損傷,以增加創傷性腦損傷病人的存活率。
英文摘要 According to the Statistics made by Department of Health in 2012, accident would rank sixth position among the ten leading causes of death in Taiwan. Traumatic brain injury (TBI) is the main reason of accident death, which induces exicitotoxicity resulted from release of glutamate, a neurotransmitter in neuro glial cells. However, the therapy of TBI is deficient in clinic. Therefore, the development of drugs for TBI is very important. In our previous study, resveratrol could reverse glutamate-induced cytotoxicity on CTX TNA2 astrocytes through supressing reactive oxygen species (ROS)/glycogen synthase kinase-3β (GSK-3β)/mitochondrial damage signaling pathway. Therefore, in this study, we aimed to investigate the protective effect of resveratrol and its derivatives on glutamate-induced cytotoxicity in astrocytes. First, using MTT assay, we observed that pre-treatment of 1 or 5 μM resveratrol, piceatannol, oxyresveratrol, rhapontigenin and pterostilbene all decreased glutamate-induced cytotoxicity on CTX TNA2 cells. Furthermore, to determine which types of cell death induced by glutamate, using flow cytometry with acridine orange stain and annexin V/PI stain to detect autophagy and apoptosis, respectively. As reveal in results, we showed that these derivatives all can reduce the percentage of glutamate-induced autophagy and apoptosis, indicating that resveratrol and its derivatives, especially rhapontigenin, may have the ability to protect the astrocytes from excitotoxicity. In addition, glutamate-induced increasing of the intracellular ROS was decreased after treatment with resveratrol or rhapontigenin as revealed by flow cytometry with DCFH-DA, HEt and DHR123 dye. Furthermore, pre-treatment of resveratrol or rhapontigenin induced the phosphorylation of Ser9-GSK-3β, and reduced mitochondrial depolarization, suggesting that resveratrol or rhapontigenin decreased glutamate-induced GSK-3β-mediated mitochondrial dysfunction. To demonstrate the involvement of AMP-activated protein kinase (AMPK), a selective inhibitor of AMPK, compound c, was employed. As showed by immunoblotting, compound c decrease the Ser9 phosphorylated levels of GSK-3β, aborgated the cytoprotective effect of reasveratrol and rhapontigenin. Taking together, these findings demonstrated that suppressing of ROS generation, activation AMPK and inhibition of GSK-3β participated in the neuroprotective of resveratrol and rhapontigenin. Furthermore, using MTT assay, we observed that rhapontigenin may have better ability to protect the astrocytes from excitotoxicity, suggesting that the hydroxyl group on B ring substituted by an methoxy group and an additional hydroxyl group on B ring may enhance the protective effect of resveratrol. It’ll provide a potential strategy for the development of a new drug for TBI and increasing the survival rate of TBI patients.
論文目次 章節目錄 1
縮寫表 2
中文摘要 4
英文摘要 6
圖表目次 8
緒論 9
實驗材料與方法 16
結果 25
討論34
未來實驗方向與展望 41
參考文獻 42
圖表 53
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