臺灣博碩士論文加值系統

噪音傷害是造成聽力損失最常見的原因之一。噪音性聽力損失的發病機制目前已知與噪音誘發耳蝸的ROS傷害有關，並且由於耳蝸內的毛細胞對於聲音刺激有更高的代謝需求，因此耳蝸比其他器官更容易受到氧化壓力的影響及傷害。藉由IL-1、TNF-α或是脂多醣 (LPS) 刺激單核球細胞及巨噬細胞後，可使單核球細胞及巨噬細胞分泌出高遷移率族蛋白1（HMGB1） ，此路徑稱為主動釋放；細胞壞死時，原本在細胞核內的HMGB1也會被表現釋放到細胞外，影響周圍的細胞產生發炎反應，此路徑稱為被動釋放。先前的研究顯示，HMGB1與TLR4產生結合後就會誘導NOX4的表現量，導致活性氧物質 (ROS) 以及活性氮物質 (RNS) 增加，並造成神經元細胞凋亡和壞死。本研究中，我們的主要目標是探討實驗動物噪音暴露後，耳蝸內HMGB1的變化、噪音性聽力損失的變化、以及活性氧物質相關蛋白之間的關聯性，初步結果顯示，耳蝸內的HMGB1在受到噪聲刺激後表現量會提高，且噪音暴露之後耳蝸HMGB1和活性氧物質的表現量呈現相關性; 此外，動物暴露噪音前先行注射anti-HMGB1 抗體，則其聽力閾值相較於對照組可減少5 - 10分貝的閾值變差。未來的研究將著眼於探討耳蝸噪音傷害後HMGB1所涉及的訊息傳遞，以進一步釐清以anti-HMGB1作為挽救耳蝸噪音傷害的治療策略之可行性。
關鍵字：噪音性聽力損失，高遷移率族蛋白1，活性氧物質

Noise injury is one of the most common causes of hearing loss. Noise- induced hearing loss (NIHL) following cochlear damage to noise exposure has been linked to a common pathogenesis involving the formation of reactive oxygen species (ROS). Cochleae are more vulnerable to oxidative stress than other organs because of the high metabolic demands of their mechanosensory hair cells in response to sound stimulation. High mobility group box 1 (HMGB1) can be actively secreted by macrophages/monocytes in response to exogenous and endogenous inflammatory stimuli such as bacterial endotoxin, TNF-α, IL-1, and IFN-γ, or passively released by necrotic cells and mediates innate and adaptive inflammatory responses to infection and injury. Previous studies have shown that HMGB1 after binding to TLR4 will induce NOX4 expression, leading to upregulation of oxidative/nitrosative stress and resulting in neuronal apoptosis and necrosis. This study thus investigated the dynamic change of HMGB1 level in cochlea after noise exposure and the association between HMGB1 expression and cochlear oxidative stress in an animal model of noise-induced hearing loss. Our preliminary results demonstrated that expression of HMGB1 was upregulated after noise exposure. The dynamic expression of HMGB1 was found to be associated with ROS production. We further demonstrated that anti-HMGB1 neutralizing antibody not only attenuates HMGB1 expression of noise-exposed cochleae but also ameliorates noise-induced hearing threshold shift of 510 dB in mice compared to control. Accordingly, future investigations will focus on the HMGB1 downstream signaling in the model of noise-exposed cochleae for revealing the significant role of HMGB1 on the therapeutic intervention of NIHL.

目錄 1
英文摘要 3
中文摘要 5
第一章 緒論 6
第一節 聽力受損及對耳蝸所產生之影響 6
第二節 耳蝸內發炎反應造成聽力受損 7
第三節 HMGB1及NF-κB路徑的機制 9
第四節 Anti-HMGB1 neutralization之應用 10
第二章 實驗動機與目的 11
第三章 材料與方法 13
第一節 實驗材料 13
一 試劑 13
二 抗體 15
三 儀器 16
第二節 實驗方法 16
一 動物模式 (Animal model) 16
二 噪音暴露 (Noise exposure) 17
三 Anti-HMGB1 antibody注射(Aantibody Injection) 17
四 聽性腦幹反應(Auditory brainstem response, ABR) 18
五 耳蝸標本製作 (Cochlea Dissection and Sample Preparation) 18
六 免疫組織化學染色玻片製作(Preparation of coating slide) 19
七 冷凍切片(Frozen section) 19
八 免疫組織化學染色 (Immunohistochemistry , IHC) 20
九 HEI-OC1細胞培養 (HEI-OC1 cells culture) 20
十 西方墨點法分析(Western Blot Analysis) 21
第四章 實驗結果 23
第一節 不同強度的噪音對CBA/CaJ小鼠聽力之影響 23
第二節 噪音暴露後耳蝸HMGB1以及ROS各不同時間點之表現 23
第三節 施打anti-HMGB1後對於噪音暴露的聽力閾值變化 25
第四節 施打anti-HMGB1的小鼠耳蝸於噪音暴露後的HMGB1以及ROS表現 26
第五章 討論 28
第一節 Anti-HMGB1 antibody neutralization對於耳蝸噪音暴露的保護效果 28
第二節 Anti-HMGB1 antibody是否減少噪音暴露後耳蝸毛細胞的死亡 30
第六章 結論與未來研究方向 32
第一節 結論 32
第二節 未來研究方向 32
第七章 參考文獻 33
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圖13 50

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