臺灣博碩士論文加值系統

隨著世界人口高齡化與老化相關疾病如神經退化性疾病人數也逐漸增加，因此近年多數研究學者對於中樞神經系統(CNS)退化性疾病尤加重視，包括：腦部外傷、中風、神經發炎及退化等病變。已知神經退化性疾病患者血腦屏障(BBB)之內皮細胞通常處於發炎狀態，但其致發炎機制尚未被詳細探討。於此，我們利用鼠腦微血管內皮細胞(bEnd.3)為細胞模式探究致發炎細胞激素IL-1及TNF-誘導細胞的發炎反應及其機轉，接著評估天然物對此致發炎反應之影響。首先，我們發現IL-1及TNF-可誘導某些發炎介質蛋白質與基因的表現，如MMP-9。前處理其相關抑制劑以探討其機轉，發現前處理活性氧分子(ROS)抑制劑可抑制此反應，推測IL-1及TNF-可透過ROS依賴性的訊號路徑誘使這些發炎蛋白的表現，其可能經由NADPH氧化酶(NOX)或粒線體依賴路徑。此外，實驗證實IL-1及TNF-可刺激細胞c-Src、EGFR、AKT及MAPKs (包括ERK1/2、p38及JNK1/2)等分子活化，而某些轉錄因子如NF-B與AP-1亦可被活化，專一性藥理抑制劑實驗指出這些訊號分子可能參與此發炎因子表現之路徑。最後，我們以免疫螢光法證明IL-1及TNF-可誘導ㄧ調節細胞間緊密連結的ZO-1蛋白組態改變，此反應可能經由MMP-9的作用所致，最終可能導致BBB瓦解。而先前研究發現許多天然物可能具有抗發炎、抗氧化或免疫增強的作用，且有較少的副作用。因而可用於預防或治療慢性炎症等相關疾病。然而，大多數天然物之效用缺乏科學證據證實。我們藉此發炎模式的評估結果發現許多天然之萃取物可抑制IL-1及TNF-誘使MMP-9等發炎蛋白表現，推測這些天然物中可能含有抗發炎的成分以保護腦部BBB不受破壞。未來，我們將進一步探究這些天然物之有效成分及其作用機轉。

As the population of the world ages, the elderly population and aging-related disorders are also increasing. Therefore, in recent years, most studies have focused on the aging-related disorders, the central nervous system (CNS) degenerative diseases particularly. These CNS disorders include traumatic brain injury, stroke, neuroinflammation, and neurodegeneration. It is known that endothelial cells of Blood-Brain-Barrier (BBB) in patients with neurodegenerative diseases usually have an inflammatory state, but the mechanisms are still unclear. Here, we use the brain microvascular endothelial cells (bEnd.3) as a cell model to investigate the inflammatory responses induction by proinflammatory factors IL-1 and TNF- and then the effects of some natural products on the responses. First, we found that both IL-1 and TNF- can induce several inflammatory proteins’ expression, including MMP-9 by regulating their gene and protein. Moreover, the pharmacological inhibitors were used to investigate the mechanisms. By pretreatment of cells with reactive oxygen species (ROS) inhibitors, we demonstrated that IL-1 and TNF- induced inflammatory proteins’ expression via a ROS-dependent signaling pathway, including NADPH oxidase (NOX) or mitochondria. We further found that IL-1 and TNF- stimulated activation of many signaling molecules, such as c-Src, EGFR, AKT, and MAPKs (ERK1/2, p38, and JNK1/2), which may be involved in the regulatory pathway by the specific pharmacological inhibitors in bEnd.3 cells. In transcriptional factors, IL-1 and TNF- stimulated activation of NF-B and AP-1. Finally, we found that IL-1 and TNF- induced the configurational change of ZO-1, as a cell tight junction protein, in bEnd.3 cells by immunofluorescence stain. The induction of MMP-9 by IL-1 and TNF- may be involved in the event. Furthermore, many previous studies have indicated that some natural products have anti-inflammatory, anti-oxidative, or immune-enhancing effects, which may have fewer side effects. Therefore, these natural products could be used for the prevention or treatment of chronic inflammation-related diseases. However, the effects of most natural products still lack scientific evidence. Here, we found some natural products can attenuate IL-1- and TNF--induced MMP-9 expression, suggesting that these natural products may contain anti-inflammatory components to protect the brain BBB from damage. We will further explore the active ingredients of these natural products and their mechanisms in the future.

長庚科技大學學位論文授權書
指導教授推薦書
論文口試委員會審定書
致謝
縮寫表...v
中文摘要...vii
英文摘要...ix
目錄...xi
圖目錄...xiv
第一章 緒論...1
第一節 腦部發炎病變之概述...2
第二節 血管內皮細胞在腦部發炎病變上的角色...5
第三節 天然物與保護或治療腦部發炎病變...6
第二章 研究動機與目的...9
第一節 研究動機...9
第二節 研究目的...9
第三章 實驗材料與方法...11
第一節 實驗材料...11
第二節 實驗方法...12
壹、細胞培養 (Cell culture)...12
貳、西方墨點法 (Western blotting)...12
參、明膠酶譜法 (Gelatin zymography)...13
肆、細胞total RNA萃取 (Cell total RNA extraction)...14
伍、反轉錄聚合酶連鎖反應 (Reverse transcription-PCR, RT-PCR)...15
陸、即時定量聚合酶連鎖反應(Real-time quantitative polymerase chain reaction,
Q-PCR )...16
柒、NADPH氧化酶活性測量(NADPH oxidase activity assay)...17
捌、胞內活性氧化物堆積測量(Intracellular ROS production)...18
玖、免疫螢光染色法 (Immunofluorescence staining)...18
拾、統計方法(Statistical analysis of data)...19
第四章 實驗結果...20
第一節 細胞激素IL-1及TNF-可誘使腦部微血管內皮細胞(bEnd.3)表現發炎相關蛋白...20
第二節 探討IL-1及TNF-引發腦部微血管內皮細胞株表現發炎蛋白的分子訊號機轉...21
第三節 探討IL-1及TNF-是否引發腦部微血管內皮細胞株之功能改變與其機轉...23
第四節 評估各種天然萃取物是否對IL-1及TNF-所引起腦部微血管內皮細胞發炎蛋白表現有
抑制的作用，並探討其作用機轉...24
第五章 結論...68
參考文獻...70

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