Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/92221
標題: 1. Develop an Analytical Platform for Rapid Detection of Polyphenol Compound Using Fullerene Based Mass Spectrometry 2. Label-free Quantitative Metabolomics of Adults-Onset Still's Disease
1. 利用富勒烯發展快速分析多酚類化合物之質譜平台 2. 非標定質譜定量技術於成人發作型史笛兒氏症之代謝體研究
作者: 張雅涵
Ya-Han Chang
關鍵字: 富勒烯;基質輔助雷射脫附游離質譜法;多酚類;成人發作型史笛兒氏症;全身性紅斑狼瘡;代謝體學;液相層析質譜法;主成分分析;生物標誌;Fullerene;MALDI;Polyphenol;AOSD;SLE;Metabolomics;LC/MS;Principal component analysis;Biomarker
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摘要: 
[論文題目一]
Matrix-assisted laser desorption / ionization time-of flight mass spectrometry, (MALDI-TOF MS) is a rapid and sensitive instrument for many biomolecules. However, there are interferences in the low-mass region (m/z of < 500) when using organic matrix, thereby it's unsuitable for the analysis of small molecules. Fullerenes carbon particles has a strong absorption in the ultraviolet region and non-interference matrix signal in the lower molecular weight region and because their superconducting and unique optical qualities which is widely applied in biomedical and photoelectric materials. In this study, fullerenes are used as the inorganic matrix for the analysis of surface-assisted laser desorption / ionization time-of flight mass spectrometry (SALDI-TOF MS), and determine its capabilities by the analysis of catechin and flavonoid small molecule. In the experiment of the effect of laser desorption, C70 are more superior to C60 in terms of signal intensity and signal-noise ratio. Hence, we speculate that C70 may provide a more sensitive result in the analysis of catechin and flavonoid. Finally, this platform applied to real samples, two fullerenes have the ability to detect flavonoids and catechin from real samples. In this study, C60 and C70 successfully applied on the analysis of tea catechin compounds in the sample and citrus flavonoids, and for most sample C70 has more excellent performance than C60. Thereby the platform establishing, C60 and C70 have demonstrated their potential to provide easy and quick detection of polyphenols, by the results of this study, we expected that can be applied to more compounds detection in the future.

[論文題目二]
Adult onset Still's disease (AOSD) and systemic lupus erythematosus (SLE) are all inflammatory disease, are characterized by lesions of various organs and can cause a variety of clinical manifestations, but SLE patients have malar rash significant clinically features than AOSD. The metabolomics is the collection of small molecules, metabolites, present in a cell, tissue, body fluids, or an organism, which aims to investigate overall metabolic activity and takes into account the genetic and environmental factors, allowing integration of the effects of these factors. Recently, the comprehensive profiling of the SLE metabolomics reveals evidence of heightened oxidative stress, inflammation, reduced energy generation, altered lipid profiles, and a prothrommbotic state. Therefore the purpose of this study is the use of metabolomics strategy to find out AOSD metabolic pathways and to explore metabolic profiles associated with inflammatory disease of SLE. Experiments were divided into two parts, we used high-flow and micro-flow LC-Q-TOF to explore change in the metabolic profiles respectively, principal component analysis (PCA) was utilized classify and reveal the differences, and t-test (p < 0.05) was performed the significant m/z changes between the model group and control group. Results demonstrated that AOSD group and SLE group compare to control group the differences identified features such as polyunsaturated fatty acids (PUFAs) , phosphatidylcholine (PC), phenylalanine and tryptophan etc. related with amino acids, fatty acids and phospholipids metabolism of the most representative. However the lipid disorders related to inflammatory diseases, cardiovascular complications and SLE patients development of atherosclerosis; amino acid metabolism is related to organ damage and source of energy, so these biomarkers and metabolic disturbance may be considered preliminary for tissue repair or energy demand in response to inflammation state.
In conclusion, PCA and other statistical software may be initially screened significant m/z and metabolite concentration levels may further identify biomarkers, but if we able to get commercial standards in the future, we can more accurately confirm these features, and further assess the unusual circumstances of physiological metabolism, help to enhance understanding of the mechanism of disease.

[論文題目一]
基質輔助雷射脫附游離飛行時間式質譜儀 (matrix-assisted laser desorption/ionization time-of flight mass spectrometry, MALDI-TOF MS) 為重要的分析儀器,對許多生物分子可提供快速且具靈敏度的鑑定分析結果,然而在分析小分子 (< 500 Da) 時,因傳統有機基質在低質荷比區會有基質本身訊號干擾,進而限制小分子分析物偵測,造成分析困難。富勒烯類 (fullerene) 的碳顆粒具有完整的結構、高純度、無毒、本身於紫外光區有強吸收力且在低分子量區較無基質訊號之干擾,並因其本身具有超導性與獨特的光學特質,目前被廣泛應用於生物醫學和光電材料上。在本研究中主要利用富勒烯作為無機基質發展表面輔助雷射脫附游離飛行時間式質譜儀 (surface-assisted laser desorption/ionization time-of flight mass spectrometry, SALDI-TOF MS),探究其分析兒茶素與類黃酮等多酚類化合物的能力。利用兩種不同的富勒烯 C60 及 C70 做為基質,進行 SALDI 分析,並同時比較此兩種富勒烯對脫附游離之效果。對大部分兒茶素與類黃酮素標準品偵測分析,發現 C70 比起 C60,無論在訊號強度或訊雜比都較 C60 優異,表示利用 C70 對於偵測多酚類化合物可提供較為靈敏之分析結果,因此嘗試將此平台應用至真實樣品,觀察兩種富勒烯鑑定真實樣品中兒茶素與類黃酮素的能力,結果發現C60 及 C70 皆可成功應用至茶葉樣品中的兒茶素化合物及柑橘中的類黃酮素進行初步定性之分析,且 C70 對部分樣品的表現也較 C60 優異,經由此平台之建立,證實 C60 及 C70 具有其潛力可提供方便且快速之多酚類檢測,期望藉由本研究之成果,於未來可應用至更多化合物檢測。

[論文題目二]
成人發作型史笛兒氏症(adult-onset Still's disease,AOSD)與全身性紅斑狼瘡 (systemic lupus erythematosus, SLE) 皆為發炎性免疫疾病,其特徵為會造成各種器官病變與多樣的臨床表現,但 SLE 於臨床上比 AOSD 多了蝴蝶斑的顯著特徵。代謝體是由小分子及存在於細胞、組織、體液與個體之代謝物所組成,其可用於探討疾病的代謝變化並考量基因與環境因素對代謝之影響,鑑於個體代謝的整合性變化,代謝體學分析可較能了解疾病相關變化。近年 SLE 的代謝體學分析結果已顯示代謝體類型可成為診斷性生物標誌,其代謝體類型可反應體內氧化壓力、發炎反應、能量供應減低、脂肪改變等,故本研究將以代謝體學策略探討 AOSD 之獨特代謝體類型並進一步與 SLE 之代謝類型作比較。實驗共分為兩大部分進行,分別以 High-flow 及 Micro-flow 液相層析四極柱飛行時間式質譜儀 (LC-Q-TOF) 的分析技術進行血清中代謝物的廣泛分析,再藉由主成分分析 (principal component analysis, PCA) 等統計軟體搭配資料庫搜尋,尋找顯著改變的代謝物 (p < 0.05),藉此作為生物標誌物 (biomarker)。結果顯示:AOSD 組 (n=32) 與 SLE 組 (n=38) 和控制組 (n=30) 比較,所鑑定到之差異特徵物如溶血磷脂醯膽鹼 (lysophosphatidylcholine, LysoPCs)、多元不飽和脂肪酸 (PUFAs)、膽鹼磷脂 (phosphatidylcholine, PCs) 及苯丙胺酸 (phenylalanine)、色胺酸 (tryptophan) 等與胺基酸、脂肪酸與磷脂質代謝最具代表性,而脂質異常與疾病之發炎、心血管併發症及 SLE 患者動脈粥狀硬化的發展等相關;胺基酸代謝異常則與器官病變及能量來源等相關,故綜合以上這些生物指標與其代謝途徑的異常初步認為可能是為了回應發炎狀態時的組織修復或能量需求。
本研究可得以下之結論,以主成分分析等統計軟體可初步篩選具差異性之變量,其代謝物水平濃度變化也可進一步找出生物標記,但若未來能獲得這些特徵物之標準品,可更加準確確認其身分,並進一步評估生理代謝機制的異常情形,有助於增進疾病產生機轉之了解。
URI: http://hdl.handle.net/11455/92221
Rights: 同意授權瀏覽/列印電子全文服務,2018-08-26起公開。
Appears in Collections:分子生物學研究所

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