無論是植物或動物，都需製造天然的抗生素以對抗外界環境的刺激。而防禦素 ( defensin ) 即是很重要的生物抗生素，其分成許多種類，可促進粒細胞( granules )、黏膜等抗菌的功能。其中，被稱為人類嗜中性胜肽 ( human neutrophil peptides, HNPs ) 的防禦素具有抗菌活性廣，且大量分布於哺乳動物的上皮及吞噬細胞中等兩大特點，普遍認為會出現於發炎或感染的人類組織之中。但是，除了發炎或感染，亦有科學家指稱防禦素具有其他的生理功能，如：做為先天性免疫或發炎的調節器，但相關說法還需更多證據加以佐證。

(一) 不同取樣方法與防禦素釋出之關聯
而為了探討防禦素於淚液層中的生理意義，首要考慮到的是，不同的取樣方法所導致的刺激，是否會引發出防禦素的釋出。防禦素會因為外在刺激而釋出，所以必須使用最低刺激性的取樣方法，而目前文獻上最常使用的方法為：Schirmer strip以及Microcapillary，前者為臨床上用來測試乾眼症的試紙，但是亦被指出其刺激性過強；後者則為現今公認最常使用的方法。因此為了監測防禦素於眼淚中的狀況，本篇研究提出四種取樣方法，每2小時取樣一次，共取樣5次，觀察防禦素於質量範圍2k-10k Da 下，隨著時間相對離子強度的變化。使用眼淚取樣方法如下：(a) Schirmer strip (b) Kimwipes (c) 以涼油誘導淚液流出 (d) 眼洗法 (Eye Flush) 等四種，在經過簡單的前處理後上機分析後，以基質輔助雷射脫附游離時間飛行質譜法進行監測。
綜合來說，我們發現無論是在正常人或是患有眼疾病人的淚液中，有高達70% 以上的淚液樣品中含有防禦素。而在這當中，防禦素的相對離子強度大於等於50% 的樣品甚至高達一半以上。此外，於一名未滿週歲的嬰兒淚液中，亦觀察到高含量的防禦素。由這些樣品中，可以看出防禦素於淚液中的角色，似乎並不僅限於做為發炎指標這麼簡單。 
(二) 防禦素於眼淚中週期性變化代表生理意義
最後選用對眼睛較低刺激性的眼洗法 ( Eye-flush ) ，探討防禦素在淚液中週期性變化及其可能之生理意義，並在後續擴大取樣數量及取樣時間。在有效樣品數38個下( 19 人)，有高達31個樣品都呈現早上 ( am 8:30 ) 防禦素相對高於下午 ( pm 5:30 ) 。並在後續針對3男4女進行24小時監測分析，發現防禦素會於凌晨1點過後開始大幅度升高，隨後逐漸遞減。根據實驗結果，可知道淚液中的防禦素除了外來病菌入侵時會作用外，它亦是一個具有時間性週期變化的胜肽。我們成功的以低刺激性的取樣方法，監測到淚液中防禦素週期性變化，間接證明其在淚液中扮演的角色，除了發炎及抗菌外，亦擔起先天性免疫的重責。

Natural antibiotics are produced as part of the innate immune response in plants and animals. Defensins are important biological antibiotics that are regulators of inflammation and play important roles in the immune response. Additionally, defensins contribute to the antimicrobial action of granulocytes and mucosal host defense in the skin against foreign bodies. Defensins are divided into many types according to their structures. One type known as human neutrophil peptides (HNPs) has a broad spectrum of antimicrobial activity and a high abundance in mammalian epithelia and granulocytes, especially in inflamed or infected tissues. This study explores the distribution and physiological mechanisms of HNPs in human tears.
In order to explore HNP distributions and physiological mechanisms in human tears, the tear sampling method was the most important concern, since HNP release is dependent on the sampling method. Schirmer strips and microcapillaries are major tear sampling methods. The former is also a method to determine whether the eye produces enough tears to keep it moist in the clinic, but causes irritation and increase fluid flow. The latter is much less invasive. In order to monitor the distribution of HNPs in human tear at different times of the day, we collected tear from healthy volunteers five times a day every 2 hours using four tear sampling methods. The tear sampling methods we chose involved the use of Schirmer strips, Kimwipes, microcapillaries, or eye flushing. The less invasive tear sampling method – eye flushing is applied. After simple pretreatment, the relative ion intensities of HNPs in human tears were monitored using MALDI-TOF/MS. In order to confirm our results, the number of samples was increased.
Our preliminary data showed that more than 70% of tear samples contain HNPs despite normal or patient status. Interestingly, tear samples from babies than a year old also had an abundance of HNPs. These results indicate that the roles of HNPs in human tears are more than just inflammation markers. 31 of 38 subjects had higher relative intensities for HNPs in the morning than in the afternoon. HNP levels in tear samples from 3 men and 4 women were monitored overnight without sleep. Analyses of these samples indicated that HNPs appeared at 1:00 am and decreased with time. Our data suggests that HNPs in human tears are regulated by the circadian clock as well as by inflammation. Using low-irritation sampling methods, we successfully monitored the relative ion intensities of HNPs in human tears at different times of the day.

論文審定書 i
誌謝 ii
中文摘要 iv
英文摘要 vi
目錄 viii
圖次 xi
表次 xvii
第 壹 章 緒論 1
1.1 前言 1
1.2 生理時鐘 2
1.3 人類淚液組成與功能 4
1.4 淚液的檢測方法 7
1.5 防禦素的發展歷史 8
1.5.1 防禦素的發現 8
1.5.2 防禦素的構型以及其抗菌機制 9
1.5.3 防禦素於人體的分布 13
1.5.4 淚液中的防禦素 15
1.6 基質輔助雷射脫附游離法 16
1.6.1 發展歷史 16
1.6.2 基質特性與游離機制 16
1.6.3 樣品製備 18
1.6.4 儀器裝置 19
1.6.5 遲滯時間的設計 20
第 貳 章 比較不同眼淚取樣方法對防禦素的影響 21
2.1 前言 21
2.2 論文目標 22
2.3 實驗部分 22
2.3.1 儀器裝置 22
2.3.2 化學試劑 22
2.3.3 眼淚樣品 23
2.3.4 眼淚取樣方法及前處理 23
2.3.5 實驗方法 25
2.4 結果與討論 26
2.4.1 重覆性探討 26
2.4.2 大量樣品以及嬰兒眼淚樣品分析 30
2.4.3 Schirmer strip 取樣 32
2.4.4 Kimwipes取樣 35
2.4.5 以涼油誘導淚液流出 39
2.4.6 眼洗法 45
2.4.7 綜合性比較 48
2.5 結論 49
第 參 章 探討防禦素於淚液中的週期性變化 50
3.1 前言 50
3.2 論文目標 50
3.3 實驗部分 50
3.3.1 儀器裝置 50
3.3.2 化學試劑 51
3.3.3 眼洗法取樣方法及前處理方式 51
3.3.4 口水取樣方法及前處理方式 51
3.3.5 實驗方法 52
3.4 結果與討論 53
3.4.1 密集性取樣 53
3.4.2 間隔性取樣 56
3.4.3 擴大取樣 59
3.4.4 熬夜24小時監測淚液中防禦素變化 62
3.4.5 口水中防禦素週期性變化 67
3.5 於其他體液中的防禦素分布 69
3.6 結論 69
第 肆 章 總結 70
第 伍 章 參考文獻 71

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