胃癌列為我國國人前十大癌症死因之一，且有逐年增長的趨勢。但胃癌的發生原因，可能來自先天遺傳因子的差異、後天生活環境及飲食習慣的不同而有所變異，因此要找到單一因子作為胃癌症臨床檢驗指標，有其困難之處。
本研究以基質輔助雷射脫附游離法結合飛行時間型質譜法 (Matrix-assisted Laser Desorption Ionization Time-of Flight Mass Spectrometry, MALDI-TOF/MS)，分析來自不同胃部疾病的胃幽門螺旋桿菌分離菌株，結合多變量統計分析 (主成份分析、群集分析與判別分析)，區分來自胃癌與非胃癌病患體內的胃幽門桿菌；此外，也利用MALDI-TOF/MS檢測上消化道 (食道癌、胃癌)、下消化道 (大腸癌) 病患之血便樣品。透過MALDI-TOF/MS偵測模式上的轉換，檢測血基質 (m/z 616) 與血紅蛋白α chain 帶正一價之訊號 (m/z 15126)。
一、 基質輔助雷射脫附游離法結合多變量統計分析研究不同胃幽門螺旋桿分離株菌
胃幽門螺旋桿菌一般潛藏於胃及十二指腸中，因其基因體的多變異性，與宿主免疫反應的不同，導致不同的胃部疾病。有鑑於此，不少相關領域之科學家試圖將胃幽門螺旋桿菌的生理狀況、生長環境及致病機制等詳加探討，以便窺探胃幽門螺旋桿菌的特性與胃部疾病之關聯性。
本篇研究所使用的菌株分別從九種上消化道疾病的病人身上分離出來，這些疾病包含慢性胃炎、胃潰瘍、十二指腸潰瘍、胃潰瘍併發十二指腸潰瘍、胃息肉、胃癌等。菌株透過萃取離心後，以基質輔助雷射脫附游離飛行式質譜儀快速分析胃幽門螺旋桿菌內的蛋白質與代謝物。
為了觀察來自不同病症的菌株與病症之間的關聯性，我們將這些質譜圖資訊結合多變量統計分析，以主成份分析 (Principal Component Analysis, PCA) 找出潛在性的生物指標，以階層式群集分析 (Hierarchical Cluster Analysis, HCA) 分析不同臨床菌株質譜圖的相似度；另一方面，使用判別分析 (Discriminant Analysis, DA) 找出38個判別因子，以判別函數運算後之數值，判別來自胃癌與非胃癌的臨床菌株。

二、 以基質輔助雷射脫附游離法分析大便中所存在之胃癌生物指標
根據衛生署100年統計資料，惡性腫瘤為國人十大死因之首。隨著現代人飲食習慣的改變，消化道病變的的機率也逐年攀升。消化道包含食道、胃、小腸與大腸。這些器官病變初期沒有任何明顯症狀，當惡化到某個程度時，會產生心窩痛、胃脹，噁心、食慾不振、全身疲勞、倦怠、體重減輕、貧血、甚至發生食道出血、胃部出血或大腸出血，並以血便方式排泄於體外。
目前對於消化道的早期篩檢包括利用糞便潛血反應早期篩檢後，搭配內視鏡進行確認結果。糞便潛血反應的檢測方式依偵測血基質 (Heme) 與血紅蛋白 (Hemoglobin) 的不同而分為化學法與免疫分析法。前者是利用試劑的顏色變化來判斷血基質的假過氧化反應 (Pseudo Peroxidase Activity)，使用方便且價格低廉，但缺點是容易受到食物的干擾，因此，受測者需在檢驗前作嚴格的飲食控制；後者則是透過單株抗體檢測血便中的血紅蛋白，抗體只與人類的血紅蛋白反應，專一性高，但缺點是操作需在低溫下進行，且價格昂貴。
本研究利用類胃液模擬出上消化道出血後，血紅蛋白分解並釋出血基質之現象以MALDI-TOF/MS進行偵測。另外，利用類胃液將血液中的血紅蛋白進行分解，混合不同體積於正常人糞便樣品中，以二次去離子水萃取、離心後，以MALDI-TOF/MS偵測糞便中存在之血紅蛋白帶正一價之訊號，與臨床免疫法比較。此部分結果顯示，以MALDI-TOF/MS偵測血紅蛋白有較低的偵測極限。
將相同萃取離心策略應到到本次收集的59個臨床糞便樣品 (分別來自19個食道癌、20個胃癌、20個大腸癌病患)，以MALDI-TOF/MS檢測血基質之結果與臨床檢驗化學法 (FOBT) 互相符合，只有少數樣本是化學法可偵測到，對應MALDI-TOF/MS則未偵測到，推測為化學法之檢測試劑會對糞便中具有氧化還原能力之物質進行變色所導致。而MALDI-TOF/MS可偵測到血基質，但臨床檢驗室尚未檢測，而無對照之結果，也會在日後請醫院補足這部分資訊。
在MALDI-TOF/MS檢測血紅蛋白之結果中，與臨床檢驗免疫法之結果亦有所出入。若以免疫法為參考指標，則MALDI- TOF/MS偵測結果有偽陰性之情形可能與糞便樣品基質干擾下，血紅蛋白與CHCA之結晶不佳所致。而偽陽性結果的產生，可能與判別之標準有關。

Chapter 1. Deciphering the toxicity of Helicobacter pylori clinical isolates from gastric diseases patients using MALDI-TOF/MS and multivariate analysis.
Helicobacter pylori (H. pyloyi) infection is associated with gastric diseases such as gastric polyp, chronic gastritis, gastric ulcer, gastric cancer, etc. In fact, most of the people infected not have the symptoms of gastric diseases due to the high degree of variability of gene with H. pyloyi and the specific immune responses of the hosts. In order to investigate the relationship between H.pylori and gastric diseases, the clinical strains of H. pylori isolated from patients from nine gastric diseases were extracted from the optimized extraction and analysis by MALDI-TOF/MS, then the high reproducible spectra were combined with multivariate statistical analysis including Principal Component Analysis (PCA), Hierarchical Cluster Analysis (HCA), Discriminant Analysis (DA) . In the result of PCA, there is no specific potential marker to discriminate the clinical strains to nine gastric diseases. In the result of HCA, the strains from different gastric diseases were clustered together means they have the similarity of the protein and metabolite. In the result of DA, the strains from gastric and non-gastric cancer were discriminanted by the discriminant function composed of thirty-eight discriminant variables in the spectra. This discriminant function would be confirmed by other clinical strains isolated from gastric diseases patients in the future and then would help to predict the the similarity of the protein and metabolite of the strains isolated from the gastric diseases patients whether gastric cancer or not.




Chapter 2. Biomarker discovery in the stools of gastric cancer patients using MALDI-TOF/MS.
According to the statistics of Republic 100 years from the Department of Health, cancer was the first of the ten lesding to death. With the modern change of eatiog habbits, gastrointestinal cancer has increased steadily. Gastrointestinal cancer accompanied occult gastrointestinal bleeding, and it is commonly detected by the fecal occult blood test (FOB). FOB including Guaiac-based fecal occult-blood test and immunochemical tests. Guaiac-based fecal occult-blood tests make use of the pseudoperoxidase activity of heme, and the reagent turns blue after oxidation by oxidants or peroxidases in the presence of an oxygen donor such as hydrogen peroxide, so it would have the potential of false-positive result. Immunochemical tests, which use antibodies detect against human hemoglobin with great sensitivity, but the tests are limited by loss of hemoglobin antigenicity at room temperature and require processing in a laboratory. In order to decrease the false-positive of detecting heme and decreasing the cost of the detection against hemoglobin in stools, in the study, we ues the distill water to extract the heme (m/z 616) and hemoglobin in stools and analysis with the reflectron and linear mode of MALDI-TOF/MS.
In this study, at first, we used the stimulated stomach acid decomposing the hemoglobin to release the heme, to stimulate the gastrointestinal bleeding. Second, we used the distill water to extract the hemoglobin in stools, and detected by the linear mode of MALDI-TOF/MS, and the detection limit of MALDI-TOF/MS against hemoglobin in stool was better than the immunochemical tests. Third, the same strategy was applied to fifty-nine patients (including nineteen esophageal cancer patients, twenty gastric cancer patients and colorectal cancer patients) stools to detect heme and hemoglobin by MALDI-TOF/MS and the results were compared with the fecal occult blood test.
In the detection of heme, MALDI-TOF/MS had not detect heme, but the Guaiac-based fecal occult-blood test had detected, it would be that the stools had the oxidants (not heme) to react the reagent. In addition, MALDI-TOF/MS had detected heme, but the Guaiac-based fecal occult-blood test had no results, those cases would be catched up in the future. In the detection of hemoglobin, using immunochemical tests to be the reference index, MALDI-TOF/MS had the false-negative result might come from the complicated matrix effect of stools, so that the hemoglobin could not form the good crystalline with matrix CHCA. The false-positive results of MALDI-TOF/MS might come from the criteria of hemoglobin signal.

目錄
論文審定書……………………………………………………………………..i
誌謝……………………………………………………………………………………ii
中文摘要………………………………………………………………………………iii
英文摘要………………………………………………………………………………vi
目錄………………………………………………………………………………..…ix
圖目錄…………………………………………………………………………...……xii
表目錄…………………………………………………………………………….....xv
附錄圖目錄……………………………………………………………………….…xvi
附錄表目錄…………………………………………………………………….…..xx

第一章、基質輔助雷射脫附游離法結合多變量統計分析研究不同胃幽門螺旋桿菌之胃癌生物指標
壹、 緒論………………………………………………………………………………1
一、 前言 …………………………………………………………………….…1
二、 細菌的分類……………………………………………………………...…2
三、 胃幽門螺旋桿菌………………………………………………………...…6
四、 游離源……………………………………………………………………...8
五、 統計軟體………………………………………………………………….13
六、 論文目標………………………………………………………………….19
貳、 實驗……………………………………………………………………………..20
一、 儀器設備…………………………………………………………..……...20
二、 化學試劑………………………………………………………………….21
三、 點樣方式………………………………………………………………….21
四、 細菌樣品來源………………………………………………………….…22
五、 實驗方法………………………………………………………………….24
六、 軟體設定……………………………………………………………….....26
參、 結果與討論………………………………………………………………….....28
一、 萃取條件探討………………………………………………………..…...28
二、 以MALDI-TOF/MS分析法鑑定標準細菌………………….......33
三、 以MALDI-TOF/MS分析法鑑定臨床菌株樣品………………………..44
肆、 結論…………………………………………………………………………..…52
伍、 參考資料……………………………………………………………………..…53

第二章、以基質輔助雷射脫附游離法分析大便中所存在之胃癌生物指標
壹、緒論…………………………………………………………………………..…56
一、前言 ………………………………………………………………...………56
二、消化道………………………………………………………………….….57
三、目前檢測消化道疾病之技術………………………………….….59
四、論文目標………………………………………………………………..…61
貳、實驗……………………………………………………………………………..62
一、 儀器裝置………………………………………………...……………..…62
二、 化學試劑………………………………………………...………………..62
三、 點樣方式………………………………………………………...……..…63
四、 血液樣品……………………………………………………….…………63
五、 糞便臨床樣品…………………...……………………………………..…63
六、 實驗流程……………………………………………………………….…64
七、 軟體設定………………………………………………………………….66
參、結果與討論……………………………………………………………….…….67
一、 模擬上消化道病患出血後其血紅蛋白被胃酸分解之實驗………….….67
二、 探討MALDI-TOF/MS偵測血便中之血紅蛋白偵測極限……………...…………………………………………….…….…….71
三、 臨床糞便樣品檢測結果………………………………..…………………73
肆、結論………………………………………………………………………………80
伍、參考資料………………………………………………………………………..82

附錄…………………………………………………………………………………..85

第一章、基質輔助雷射脫附游離法結合多變量統計分析研究不同胃幽門螺旋桿菌之胃癌生物指標
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第二章、以基質輔助雷射脫附游離法分析大便中所存在之胃癌生物指標

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