臺灣博碩士論文加值系統

咖啡因可能有助於緩解乾眼症是一項重要的課題，最近一項研究發現攝取咖啡因者罹患乾眼症的盛行率是13%，然而無攝取者的盛行率則是16.6%，另外近期有Arita et al. (2012) 與Osei et al. (2014) 兩篇小型臨床試驗的論文指出攝食咖啡因會增加淚液分泌量，但都缺少咖啡因促使淚液短期分泌的作用機制探討。因此本研究以小鼠in vivo與小鼠外淚腺ex vivo實驗分析咖啡因對淚液分泌的影響，並探討咖啡因作用於外淚腺P1和P2 purinergic receptors促進淚液短期分泌之路徑及其動態機制變化。In vivo實驗結果發現：一、ICR母鼠的外淚腺在10週齡時已足夠成熟，可對咖啡因的影響產生反應；二、20 mg/kg與40 mg/kg的咖啡因劑量在管餵後45與60分鐘時淚液量皆有顯著的增加，以40 mg/kg咖啡因劑量管餵45分鐘後的淚液分泌最多；三、在咖啡因的影響下，外淚腺中P1 receptor與P2 receptor皆有正向調控 (upregulation) 的現象。Ex vivo實驗結果發現在咖啡因的短期影響下，咖啡因主要是藉由淚腺中的P1 receptor促進淚液分泌，在P1和P2 receptors雙重抑制狀況下，交感與副交感神經末梢對於較長時間咖啡因的影響產生負回饋的效果，進而抑制淚液的分泌。本研究提供以咖啡因促進淚液分泌的基礎研究結果，將在未來幫助研發緩解乾眼症的新型藥物。

The potential of caffeine in alleviating dry eye syndrome has emerged as a notable topic in recent years. Recent research showed that the prevalence of dry eye syndrome among caffeine consumers is 13%, compared to 16.6% in non-caffeine consumers. Another two small-scale clinical trials by Arita et al. (2012) and Osei et al. (2014) indicated that caffeine intake increased tear secretion, yet the short-term mechanism behind this effect was not explained. This study investigated the effects of caffeine on tear secretion with in vivo experiments on mice and ex vivo experiments on mouse extraorbital lacrimal glands. The caffeine effects on pathways through of P1 and P2 purinergic receptors in the lacrimal glands and the dynamic mechanism were examined. The in vivo results revealed: 1. Extraorbital lacrimal glands of ICR female mice are sufficiently mature by 10 weeks of age to respond to caffeine. 2. Significant tear secretion increase was observed at 45 and 60 minutes after oral caffeine administration at 20 mg/kg and 40 mg/kg, with the highest secretion found at 45 minutes after 40 mg/kg caffeine administration. 3. Both lacrimal gland P1 and P2 receptors were upregulated after receiving caffeine. The ex vivo experiments demonstrated that caffeine primarily promotes tear secretion through the P1 receptor in the extraorbital lacrimal gland. Under dual inhibition of P1 and P2 receptors, a negative feedback effect from sympathetic and parasympathetic nerve endings was observed and tear secretion was inhibited. This study has provided the essential data on caffeine-induced tear secretion and contributed to lay up the foundation in developing novel pharmaceutics for dry eye syndrome.

中文摘要 I
英文摘要 II
表目錄 V
研究動機 1
第一章 文獻回顧 6
第一節 淚液膜與外淚腺 (Tear film and extraorbital lacrimal gland) 6
第二節 嘌呤受體 (Purinergic receptors) 11
第三節 咖啡因 (Caffeine) 13
第四節 研究問題 15
第二章 實驗材料與方法 16
第一節 實驗設計 16
第二節 實驗動物 21
第三節 實驗方法 22
第三章 實驗結果 31
第一節 淚腺成熟度對於咖啡因的反應以及咖啡因促進淚液分泌的時間點 31
第二節 咖啡因在小鼠模式中劑量與時間影響淚液分泌的效應 33
第三節 咖啡因在小鼠模式中劑量與時間影響purinergic receptors之活化情形 35
第四節 咖啡因在小鼠外淚腺體外培養中調控purinergic receptors路徑影響淚腺蛋白分泌之效應 43
第五節 咖啡因在小鼠外淚腺類器官中調控P1與P2 purinergic receptors路徑影響淚液分泌之效應 45
第四章 討論 57
第五章 參考文獻 65
第六章 附錄 70
表目錄
圖 1 以五週大和十週大ICR母鼠（n = 3)管餵20 mg/kg咖啡因後的10-100 分鐘各時間點與淚液分泌基準量相比之淚液分泌平均變化量百分比 31
圖 2 以十週大ICR母鼠（n = 6）依組別餵食20 mg/kg和40 mg/kg咖啡因後，分別測試45或60分鐘後的淚液分泌量與淚液分泌基準量相比之淚液分泌平均變化量百分比 33
圖 3 以免疫組織化學染色法顯示在不同組別的外淚腺中A1 receptors的表現位置與情形 (400×) 36
圖 4 以免疫組織化學染色法顯示在不同組別的外淚腺中A2A receptors的表現位置與情形 (400×) 37
圖 5 以免疫組織化學染色法顯示在不同組別的外淚腺中A2B receptors的表現位置與情形 (400×) 38
圖 6 以免疫組織化學染色法顯示在不同組別的外淚腺中P2X7 receptors的表現位置與情形 (400×) 39
圖 7 以免疫組織化學染色法顯示在不同組別的外淚腺中P2Y2 receptors的表現位置與情形 (400×) 40
圖 8 以western blot結果定量在在不同組別的外淚腺中A1、A2A、A2B、P2X7以及P2Y2 receptors的表現量。 41
圖 9 以外淚腺體外培養觀察在去除交感與副交感神經的影響下，咖啡因介入後外淚腺的蛋白分泌情形 43
圖 10 以類器官膨脹試驗 (organoid swelling test) 觀察在各組別咖啡因與不同受體抑制劑介入後第1小時與第4小時的類器官相對於第0小時 (t0) 的面積變化倍率 45
圖 11 類器官膨脹試驗 (organoid swelling test) 中，各組的類器官在咖啡因與不同受體抑制劑影響下第0、1、4小時的面積變化照片 48
圖 12 類器官膨脹試驗 (organoid swelling test) 中，Blank組的類器官在第0-4小時之面積變化照片 (40×) 49
圖 13 類器官膨脹試驗 (organoid swelling test) 中，Forskolin組的類器官在Forskolin影響下第0-4小時之面積變化照片 (40×) 50
圖 14 類器官膨脹試驗 (organoid swelling test) 中，Caffeine 0.3mM組的類器官在咖啡因影響下第0-4小時之面積變化照片 (40×) 51
圖 15 類器官膨脹試驗 (organoid swelling test) 中，Caff + block p/sR組的類器官在咖啡因與phentolamine、propranolol、scopolamine影響下第0-4小時之面積變化照片 (40×) 52
圖 16 類器官膨脹試驗 (organoid swelling test) 中，Caff + block P1R& p/sR組的類器官在咖啡因與MRS1523、phentolamine、propranolol、scopolamine影響下第0-4小時之面積變化照片 (40×) 53
圖 17 類器官膨脹試驗 (organoid swelling test) 中，Caff + block P2R& p/sR組的類器官在咖啡因與suramin、phentolamine、propranolol、scopolamine影響下第0-4小時之面積變化照片 (40×) 54
圖 18 類器官膨脹試驗 (organoid swelling test) 中，Caff + block P1, P2R組的類器官在咖啡因與MRS1523、suramin影響下第0-4小時之面積變化照片 (40×) 55
圖 19 類器官膨脹試驗 (organoid swelling test) 中，Caff + block P1, P2R & p/sR組的類器官在咖啡因與MRS1523、suramin、phentolamine、propranolol、scopolamine影響下第0-4小時之面積變化照片 (40×) 56

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