類風濕性關節炎模式小鼠之唾液蛋白質體與 盲腸、糞便菌相分析;Analysis of saliva proteome and cecum/ fecal bacteria community in rheumatoid arthritis model mice

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Title:	類風濕性關節炎模式小鼠之唾液蛋白質體與盲腸、糞便菌相分析;Analysis of saliva proteome and cecum/ fecal bacteria community in rheumatoid arthritis model mice
Authors:	張筑喻;Chang,Chu-Yu
Contributors:	生命科學系
Keywords:	類風溼性關節炎;唾液;蛋白質;菌相;Rheumatoid arthritis;Saliva;Protein;bacteria community
Date:	2016-08-24
Issue Date:	2016-10-13 13:04:24 (UTC+8)
Publisher:	國立中央大學
Abstract:	類風濕關節炎（RA）是一種複雜的慢性，全身性自身免疫性疾病。它會影響關節、骨骼進而促進關節破壞或者是骨骼畸形。儘管RA的研究已經完成多年，該疾病的發病機制目前仍較少被了解而使其難以幫助診斷。建立實驗模式動物可以幫助研究，並且了解疾病病源機制或是治療。在本論文，我們的研究目的為以人類試驗為目標，藉此建立在類風溼性關節炎之蛋白質體學與微生物菌相分析平台，並希望找出與疾病之關聯性。因此，我們以類風溼性關節炎模式動物為研究樣本並分析其唾液、血清蛋白質體與口腔黏膜、盲腸黏膜、盲腸內容物及糞便之微生物菌相。首先，利用基質輔助雷射脫附離子化－飛行時間質譜儀 (MALDI TOF MS) 分析健康與疾病之小鼠唾液的不同表現蛋白質圖譜。根據結果發現，分子量約1418 (Da)只出現在健康樣本中，2702 (Da)則只出現在疾病之樣本。m/z 在6370、6995、11285、13979以及22655在健康與疾病之樣本皆有峰值，但其相對含量則是在疾病樣本中逐漸變低。其次，SDS-PAGE電泳分析血清之控制組及疾病蛋白質體並沒有顯著差異。最後，在盲腸黏膜與內容物之樣本，Parabacteroides merdae在RA第一週佔了約70%；另一方面，Akkermansia muciniphila在RA第四週佔了約70%。結論，利用MALDI-TOF MS能快速分析唾液蛋白質圖譜。在血清樣本中沒有顯著差異之蛋白質體，但仍然發現在發炎反應持續進行時，白蛋白在疾病樣本與健康之間其相對含量減少，此情形在RA之患者中已被證實存在。 A. muciniphila 與體重減輕相關並且期會增厚腸道黏膜並預防發炎反應。在本結果中，推測在RA第四週，關節炎指數最高週數，A. muciniphila 會影響小鼠體重並使其減輕。而P. merdae 則是在腸道發炎環境下會較少含量，與本結果中對照，在RA第四週之樣本中P. merdae之含量與RA第一週有明顯減少趨勢。希望未來能繼續藉由動物模式了解類風溼性關節炎與蛋白質學及微生物菌相之間的關係，能幫助未來在人體臨床上之治療與診斷，以達到分析平台之效用。;Rheumatoid arthritis (RA) is a complex, chronic, and systemic autoimmune disease. It affects joints, and bones, promoting joints destruction as well as bone deformities. Although researches of RA has been ongoing for many years, still relatively less understanding of its pathogenesis were known and it is difficult to diagnosis. Established animal models can assists in the research progress. It helps to understand the disease pathogenesis or treatment. In this thesis, our aim is to establish platform to analyze proteome, bacterial community in RA, finding the relationship with disease and do for future experiment of human. Therefore, we chose animal models of RA for analysis of saliva, serum proteome and oral mucosa, cecum mucosa, cecum content and stool for bacterial community. First, the differentially expressed protein profiles in the saliva between healthy and RA disease mice were analyzed by matrix assisted laser desorption/ionization time- of –flight mass spectrometry (MALDI- TOF MS). According to the results, we found the peak of m/z 1418 was only appearing in healthy control, but not in disease sample, and the peak of m/z at 2702 was only appearing in disease samples not in healthy sample, relatively. Peak of m/z near 6370, 6995, 11285, 12728, 13979 and 22655 were show in both healthy and disease samples. However, the intensities were low in disease samples. Second, we found between healthy and disease serum that there was no significant difference by SDS-PAGE gel in mice serum protein. Finally, Parabacteroides merdae was among 70% in RA 1-week, on the other hand, Akkermansia muciniphila was among 70% in RA 4-week mice cecum mucosa and content samples. The conclusion is that saliva protein profiling by MALDI-TOF MS is a quickly method. And even though there was no significant difference in serum protein, albumin still had low intensity in disease sample when the inflammation was continued and it confirm in RA patients. For A. muciniphila it was related to weight loss and can increase intestinal mucosa thickness to prevent inflammation. In our result we surmise in RA 4-week, which is of high arthritis score and the A. muciniphila was affecting mice weight. P. merdae decreased in inflammation intestinal environment and in our result the RA 4-week that P. merdae was low than RA-1 week. We hope that in the future we will continue to use animal model of rheumatoid arthritis and understand the relationship between proteome, microbiome and disease. And can help for the clinical treatment and diagnosis in humans in the future, in order to complete the analysis platform.
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