開發鈉鉀離子交換系統應用於體外耳蝸前驅細胞分化

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姓名

丁郁軒(Yu-Hsuan Ting) 查詢紙本館藏

畢業系所

生物醫學工程研究所

論文名稱

開發鈉鉀離子交換系統應用於體外耳蝸前驅細胞分化
(To establish Sodium/Potassium Exchange System for In-vitro Cochlear Progenitor Cell Differentiation)

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至系統瀏覽論文 (2028-1-31以後開放)

摘要(中)

聽力損失是一種常見病，會給生活帶來諸多不便。導致聽力損傷的因素有很多，如遺傳缺陷、環境噪聲、耳毒性藥物和衰老的影響。隨著人口老齡化問題，聽力損失人數將從2019年的1.5億增加到2050年的2.5億。聽力損失可分為兩種類型。第一種是傳導性聽力損失，通常是由於耳道內有異物阻塞或中耳腔積液所致。臨床上常採用手術治療或助聽器輔助來放大聲音訊號。另一種是感音神經性聽力損失（SNHL），多數是由於毛細胞損傷後無法再生，導致神經信號無法進入大腦，目前臨床上常見的治療方法是植入人工電子耳，但是成本很高而且不能根治。因此，引入細胞治療聽力損失是一種有潛力的方法。從胚胎發育的角度，我們知道在毛細胞分化的早期，內淋巴液從高鈉緩慢地變為高鉀。本研究的目的是利用海藻酸鉀加入氯化鍶交聯形成具有半透膜的K-beads。利用鈉鉀離子對海藻酸鹽的親和力不同，使培養基中的鈉離子被海藻酸鹽吸附。因此，K-beads 可以釋放鉀離子並減少鈉離子，從而創造動態離子調節，形成毛細胞分化的體外微環境。我們成功開發了一種體外動態離子調節系統，用於模擬體內內淋巴微環境變化。

摘要(英)

Hearing loss is a common disease that can cause many inconveniences in life. There are many factors that can cause hearing loss, such as genetic defects, environmental noise, ototoxic drugs, and the effects of aging. With the ageing population problem, the number of people with hearing loss will increase from 150 million in 2019 to 250 million in 2050.Hearing loss can be divided into two types. The first is conductive hearing loss, which is usually caused by foreign body obstruction in the ear canal or fluid accumulation in the middle ear cavity. Clinically, surgical treatment or hearing aid assistance is often used to amplify sound signal. The other one is sensorineural hearing loss (SNHL), most of which is due to the damage of hair cells without regeneration, resulting in the inability of nerve signals to enter the brain At present, the common clinical treatment is to place the cochlear implant, but the cost is very high but cannot cure. Therefore, introducing cell therapy for hearing loss is a potential method. From the point of view of embryonic development, we knew the endolymphatic fluid changes from high sodium to high potassium slowly during the early stage of hair cell differentiation. The aim of the study, we use potassium alginate to add strontium chloride to cross-link to form K-beads with a semi-permeable membrane. Consequently, the K-beads can release potassium ions and reduce sodium ions to create dynamic ion regulation forming in-vitro microenvironment for hair cell differentiation. In summary, we successfully developed in-vitro dynamic ion regulation system for mimicking in-vivo endolymphatic microenvironment.

關鍵字(中)

★ 聽損
★ 毛細胞
★ 內淋巴液
★ 海藻酸鉀

關鍵字(英)

★ hearing loss
★ hair cell
★ endolymph
★ potassium alginate

論文目次

一、研究背景暨文獻回顧 1
1-1 耳部構造及功能 1
1-1-1 構造 1
1-1-2 聽覺傳導 2
1-1-3 內淋巴液 3
1-1-4 聽毛細胞 5
1-1-5 內耳發育 7
1-2 聽力損失 9
1-2-1 聽力損失介紹 9
1-2-2 聽力損失原因 10
1-2-3 聽力損失類型 11
1-2-4 聽力損失影響 11
1-3 現行治療方式 12
1-3-1 手術 12
1-3-2 助聽器 12
1-3-3 人工電子耳 13
1-3-4 聽毛細胞再生 14
1-4 再生醫學 15
1-4-1 細胞治療 15
1-4-2 組織工程 15
1-4-3 三維細胞培養 17
1-4-4 耳蝸前驅細胞 18
1-5 海藻酸鹽(alginate) 19
1-5-1 細胞支架(scaffold) 19
1-5-2 海藻酸鹽應用 20
1-6 海藻酸鉀 20
1-6-1 海藻酸鉀特性 20
1-6-2 二價陽離子 21
1-6-3 海藻酸鹽類可用來去除水中含氮物質 22
二、研究動機與目的 23
25
三、實驗藥品與實驗方法 26
3-1 實驗藥品與器材 26
3-1-1 藥品清單 26
3-1-2 設備清單 27
3-1-3 縮寫對照表 28
3-2 細胞支架設計 29
3-2-1 製作海藻酸鹽細胞支架 29
3-2-2 添加鉀鹽之海藻酸鈉支架 30
3-3 細胞支架特性分析 31
3-3-1 掃描式電子顯微鏡 (Scanning Electron Microscopy, SEM) 31
3-3-2 傅立葉變換紅外光譜 (FT-IR) 31
3-3-3 鈉鉀離子濃度置換 32
3-4 海藻酸鉀水凝膠球體 33
3-4-1 海藻酸鉀溶液滅菌 33
3-4-2 海藻酸鉀球體製備 33
3-4-3 利用海藻酸鉀球體改變鈉鉀離子濃度 33
3-4-4 海藻酸鉀球體保存測試 34
3-4-5 生物相容性測試 34
3-5 細胞培養 35
3-5-1 Pre-plate 35
3-5-2 細胞存活及死亡染色 36
四、結果與討論 37
4-1 海藻酸鈉支架 37
4-1-1 海藻酸鈉支架製備 37
4-1-2 海藻酸鈉支架外觀 40
4-1-3 海藻酸鈉支架穩定性 41
4-1-4 海藻酸鈉支架結構 42
4-1-5 磷酸二氫鉀-海藻酸鈉支架成分分析 43
4-1-6 培養基離子濃度 44
4-2 海藻酸鉀球體 45
4-2-1 海藻酸鉀球體外觀 45
4-2-2 不同滅菌方式對離子濃度影響 46
4-2-3 不同比例海藻酸鉀球體離子置換效果 47
4-2-4 模擬耳蝸成熟離子濃度變化 48
4-2-5 海藻酸鉀球體保存 50
4-2-6 海藻酸鉀球體去除NH3/NH4+效果 51
4-2-7 動態迴流系統 52
4-2-8 生物相容性測試 53
4-2-9 耳蝸前驅細胞生長及分化 54
4-2-10 耳蝸細胞團塊在不同離子濃度下尺寸變化 56
4-2-11 耳蝸前驅細胞Live & Death染色 57
五、結論 58
六、參考文獻 59

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指導教授

陳靖昀(Ching-Yun Chen)

審核日期

2023-2-1

推文