臺灣博碩士論文加值系統

圓窗膜為介於中耳及內耳間的膜狀結構，是治療性藥物從中耳腔進入內耳的主要路徑。藥物局部送入內耳包含兩種方式：intratympanic以及intracochlear approaches。Intratympanic approach就是透過圓窗膜將藥物輸送進入內耳。近年來，微氣泡超音波已廣泛使用在影像造影及藥物、基因的輸送，本研究目的在於研究微氣泡超音波是否能提升圓窗膜的通透性，以促進藥物自中耳腔輸送進入內耳。本實驗室使用氣體為核心，外覆白蛋白之微氣泡。以天竺鼠做為實驗動物模式，讓其鼓泡（tympanic bulla）充滿微氣泡及biotin–fluorescein isothiocyanate conjugates（biotin-FITC）的混合物，在不同的試驗條件下（包括不同超音波作用時間的實驗組或是單純浸泡藥物於圓窗的控制組），評估微氣泡超音波將biotin-FITC輸送入內耳的濃度。此外，我們使用Alexa Fluor 488-conjugated phalloidin做為追蹤的tracer，觀察微氣泡超音波作用在圓窗膜後所產生的效應。我們也使用gentamicin-Texas Red conjugates 以及gentamicin免疫螢光的實驗方式來評估內耳毛細胞uptake此aminoglycosides類藥物的情況。期間，以聽性腦幹反應檢查來評估微氣泡超音波是否造成動物聽力閾值的改變。研究結果顯示，相較於單純浸泡圓窗的控制組，微氣泡超音波可有效增加biotin-FITC進入內耳的效率約3.5到38倍。以Alexa Fluor 488-conjugated phalloidin做為tracer，可以看到圓窗膜在微氣泡超音波作用後立即增加其通透度; 內耳毛細胞對gentamicin的攝入也因微氣泡超音波的作用下而顯著提升; 聽力閾值於微氣泡超音波作用後並無顯著改變。我們驗證微氣泡超音波作用於圓窗膜可明顯提升內耳的藥物輸送，提供未來臨床內耳藥物輸送治療策略的重要參考依據。目前正在進行研究之初步結果顯示微氣泡超音波能幫助類固醇藥物dexamethasone及抗氧化劑N-acetylcysteine局部輸送至內耳，因而提升藥物降低噪音性聽損之效用。

The round window membrane (RWM) acts as a barrier between the middle ear and cochlea and can serve as a crucial route for therapeutic medications entering the inner ear via middle ear applications. The approach in local delivery of drugs to inner ear includes intratympanic and intracochlear approach. The intratympanic approach has the advantage of no insult to inner ear structure and function in comparison of the intracochlear approach. Drug delivery via intratympanic approach depends on the permeation of round window membrane. Microbubbles (MBs) ultrasound is a promising technique that can promote drug delivery but was never applied to inner ear drug delivery. In this study, we targeted the practical application of MB ultrasound on increasing the RWM permeability for facilitating drug or medication delivery to the inner ear. Using biotin–fluorescein isothiocyanate conjugates (biotin–FITC) as delivery agents and guinea pig animal models, we showed that MB ultrasound exposure can improve the inner ear system use of biotin–FITC delivery via the RWM by approximately 3.5 to 38 times that of solely soaking biotin–FITC around the RWM for spontaneous diffusion. We also showed that there was significant enhancement of hair cell uptake of gentamicin in animals whose tympanic bullas were soaked with MB-mixed gentamicin–Texas Red or gentamicin and exposed to ultrasound. Furthermore, increased permeability of the RWM from acoustic cavitation of MBs could also be visualized immediately following ultrasound exposure by using Alexa Fluor 488-conjugated phalloidin as a tracer. Most importantly, such applications had no resulting damage to the integrity of the RWM or deterioration of the hearing thresholds assessed by auditory brainstem responses. The findings provide a basis for MB ultrasound-mediated techniques with therapeutic medication delivery to the inner ear for future application in humans. The next issues of experiments are to investigate the benefit of local drug delivery from MB ultrasound in the animal model of inner ear disease. The preliminary results in the ongoing experiments revealed this technique could promote dexamethasone and N-acetylcysteine delivering to inner ear and thus enhance the drug action to ameliorate noise-induced hearing loss.

第一章、摘要… 1
第二章、緒論… 5
第三章、目的…15
第四章、材料與方法…17
第一節、實驗動物與研究設計…17
第二節、微氣泡以及輸送至內耳試劑的準備…18
第三節、Gentamicin與Texas Red的結合…18
第四節、超音波作用…19
第五節、手術步驟…19
第六節、Biotin-FITC螢光強度的量測…20
第七節、樣本準備與收集…21
第八節、免疫螢光染色…21
第九節、共軛焦顯微鏡檢…22
第十節、聽性腦幹反應檢查…23
第五章、結果…24
第一節、微氣泡超音波可促進biotin-FITC從中耳腔輸送至內耳…24
第二節、微氣泡超音波的蝕穴效應可增加圓窗膜通透性…25
第三節、微氣泡超音波作用後圓窗膜通透性的變化…26
第四節、不同模式微氣泡超音波作用可以提升內耳藥物輸送效率高達3.5到38倍…27
第五節、微氣泡超音波可促進gentamicin從中耳腔輸送至內耳…28
第六節、微氣泡超音波的作用不會傷害聽力…29
第六章、討論…39
第七章、結論…43
第八章、目前實驗結果及後續實驗方向…44
第九章、參考文獻…51
附錄一、刊載於“Journal of Controlled Release”雜誌全文…59

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