臺灣博碩士論文加值系統

噪音傷害會藉由聚集周邊的白血球的作用來引起耳蝸組織的發炎反應。然而，參與發炎反應的細胞角色以及相關的細胞激素(cytokines)、趨化細胞激素(chemokines)的變化，均有待進一步釐清。美滿黴素(minocycline)為第二代四環黴素(tetracycline) 的製劑，不僅臨床上常用來作為對抗微生物感染的抗生素，它所具備的抗發炎與減少缺血性(ischemia)組織傷害的效用，已廣泛被應用於退行性神經性損傷、腦缺血、類風濕性關節炎、多發性硬化症的預防與治療研究。由於缺血性傷害與炎症反應均是噪音性的聽損的重要致病機制，因此我們也將針對minocycline是否能夠對噪音傷害提供保護性的角色加以探討。為了證明這個假說，我們先將CBA/CaJ小鼠暴露在115分貝的寬頻噪音，一天三小時，分別抽取未受噪音之對照組、以及噪音傷害一天及連續三天之實驗組小鼠耳蝸中的淋巴液，以 Bio-Plex Suspension Assay進行細胞激素與趨化細胞激素的變化分析。暴露噪音的小鼠耳蝸淋巴液中，RANTES、MIP-2、IL-12(p40)、IL-6、IL-1α與MCP-1表現增加。噪音暴露後的不同時段，取下注射minocycline及未注射minocycline的耳蝸，利用反轉錄酶聚合酶連鎖反應方法(RT- PCR)，進行細胞激素與趨化細胞激素的轉錄變化分析，IL-6, MCP-1 and RANTES在噪音後的立即(0小時)、3小時、24小時均呈現轉錄增加，minocycline的作用則會使RANTES的表現減少。製作耳蝸標本進行組織免疫化學染色(immunohistochemistry)，利用F4/80及CD45偵測相關的白血球表現。在噪音暴露之後24小時，F4/80及CD45的表現明顯增加，並在minocycline的作用之後明顯減少。以聽性腦幹反應(auditory brainstem response)測量小鼠聽力，在暴露噪音之後，經minocycline注射的小鼠聽力閾值波動小於未注射minocycline，這些結果表示了minocycline在噪音暴露的耳蝸模式中具有抗發炎及預防聽力損傷的作用。

A cochlear inflammatllory response has been demonstrated in response to acoustic trauma by recruiting circulating leukocytes. However, the role of involved chemokines and cytokines remains to be further clarified. Minocycline, a semisynthetic second generation tetracycline, is a well known broad spectrum antibiotic. It has been shown to possess remarkable anti-inflammatory and neuroprotective properties in models of neurodegeneration, brain ischemia, rheumatoid arthritis and multiple sclerosis. We thus hypothesize that minocycline may also target part of the inflammatory responses in noise-exposed cochlea. To verify this hypothesis, CBA/CaJ mice were exposed to white band noise, 115 dB SPL, 3 hr per day for 3 days. Cochlear fluid obtained from noise-exposed and control groups were analyzed by determining the level of cytokines and chemokines using Bio-Plex Suspension Assay. We observed that RANTES、MIP-2、IL-12(p40)、IL-6、IL-1α、and MCP-1 were overexpressed in noise-exposed group. Cochlear obtained from minocycline treatment group and control animals after noise exposure were analyzed to determine the differential transcriptional levels of noise-related chemokines /cytokines. The result showed that genes of IL-6, MCP-1 and RANTES in noise-exposed cochlea were up-regulated during a 24-hr time course. Minocycline was found to significantly decrease RANTES at transcriptional level. Leukocytes were identified immunohistochemically with antibodies to CD45 and F4/80. In control cochleae, the number of CD45- and F4/80-positive cells was dramatically increased 24 hr after noise trauma; while leukocytes recruitment was significantly decreased in minocycline treatment group. Auditory brainstem responses revealed much less post-exposure threshold shift in minocycline group than that in control group. These data suggest that minocycline potentially play an anti-inflammatory role and prevent hearing deterioration in a noise-exposed cochlea model.

目錄 I
英文摘要 IV
中文摘要 VI
第一章、緒論 1
第一節、聽力損傷及對耳蝸產生的影響 1
第二節、耳蝸中的發炎反應及其途徑 3
第三節、噪音傷害在耳蝸造成的發炎反應 4
第四節、Minocycline的介紹及其作用 5
第二章、實驗動機與目的 8
第三章、材料與方法 9
第一節、實驗材料 9
壹、試劑 9
貳、抗體 11
第二節、實驗方法 11
壹、動物模式 (Animal model) 11
貳、噪音暴露(Noise exposure) 12
参、Minocycline 注射 12
肆、聽性腦幹反應 (Auditory brainstem respose, ABR) 12
伍、耳蝸標本的製作 (Cochlea dissection and sample preparation) 13
陸、免疫組織化學染色玻片製作 (Preparation of coating slide) 14
柒、冰凍切片(Frozen section) 14
捌、免疫組織化學染色 (Immunohistochemistry, IHC) 15
玖、RNA萃取 (RNA extraction) 15
拾、RNA反轉錄 (Reverse Transcription) 17
拾壹、聚合脢連鎖反應 (Polymerase chain reaction, PCR) 18
拾貳、耳蝸表面處理(Surface preparation) 19
拾参、Bio-Plex suspension assay 19
拾肆、HEI-OC1細胞培養 (HEI-OC1 cells culture) 23
拾伍、WST-1細胞增生分析 (WST-1 Cell Proliferation Assay) 24
拾陸、統計分析 (Statistical analysis) 25
第四章 實驗結果 26
第一節、噪音暴露之後耳蝸淋巴液細胞激素與趨化激素的表現 26
第二節、Minocycline對耳蝸細胞株生長效應及對耳蝸組織可能的保護作用 26
第三節、Minocycline減少低氧環境以及噪音引起的細胞激素與趨化激素 27
第四節、Minocycline 會減少噪音引起的發炎細胞表現 29
第五節、Minocycline 減少噪音引起的毛細胞傷害 30
第六節、Minocycline會減少噪音引起的聽力損傷 31
第五章、討論 33
第一節、Minocycline對耳蝸細胞與實驗動物的影響 33
第二節、噪音引起的發炎反應 34
壹、細胞激素及趨化激素在噪音暴露後的表現 34
貳、耳蝸發炎細胞在噪音暴露後之表現 36
第三節、噪音對聽力的損傷 36
第四節、Minocycline在噪音傷害後的保護效果 37
第六章、結論與未來研究方向 39
第一節、結論 39
第二節、未來研究方向 39
第七章、參考文獻 41

圖目錄
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圖2 46
圖4 48
圖5 49
圖6 50
圖7 51
圖8 52
圖9 53
圖10 54

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