博碩士論文 103821010 詳細資訊




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姓名 馮健榮(Jian-Jung Feng)  查詢紙本館藏   畢業系所 生命科學系
論文名稱 建立人類皮膚益生微生物菌組銀行
(Establishment of a human skin probiotic microbiome bank)
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摘要(中) 人類皮膚微生物菌相失衡已被證實與許多皮膚疾病有關。目前主要分析皮膚上菌相的方法為價格昂貴的次世代定序。我們已經從皮膚上分離許多細菌並用16S rRNA序列來鑑定出其學名,並經由實驗找出其具有利用多種不同碳源進行發酵的活性。這些具有發酵活性的細菌我們定義其為益生菌,並儲存在我們的實驗室作為”皮膚益生菌銀行”。為了定量人體血液中自然產生之皮膚菌抗體,我們已經開發出一種免疫色層快篩試紙:由吸收墊、硝化纖維膜、結合上 Protein A 的奈米金和樣品墊組成。在硝化纖維膜上有實驗組區和控制組區分別點上細菌加熱破膜菌液和抗人類免疫球蛋白G二級抗體。在人類血液中,我們已成功使用免疫色層快篩試紙偵測到在皮膚菌中數量最多的痤瘡桿菌之抗體,顯示這種快篩試紙半定量人類皮膚微生物菌群抗體的可行性。
摘要(英) The dysbiosis of the human skin microbiome has been associated with many skin disorders. The next generation sequencing (NGS), although it is cost-effective, is a major method to analyze the dysbiotic skin microbiome. We have isolated many skin bacteria, identified them by 16S rRNA sequencing and tested their fermentation activities using various carbon sources. The fermenting bacteria are defined as probiotic bacteria and stored in our laboratory as a “Skin Probiotic Bank”. To quantify the antibodies to skin bacteria naturally produced in human blood, we have contracted an immunochromatographic test strip consisting of absorbent pad, nitrocellulose (NC) membrane, protein A -conjugated gold nanoparticle pad and sample pad. Test and control zones on NC membranes are created by spotting the heat-killed lysates of bacteria and anti-human IgG secondary antibody. In the human blood, we are able to detect the antibodies to Propionibacterium acnes, a most abundant bacterium in human skin, using immunochromatographic test strips, demonstrating the feasibility of the test strips for semi-quantitatively profiling the natural antibodies to the huamn skin microbiome.
關鍵字(中) ★ 微生物總體
★ 益生菌
★ 慢性腎臟病
★ 痤瘡
關鍵字(英) ★ Microbiome
★ Probiotic
★ Chronic renal disease
★ Acne
論文目次 目錄
中文摘要 i
Abstract ii
謝誌 iii
目錄 iv
圖目錄 vi
表目錄 vii
第壹章、前言 1
1.1 人類微生物總體與疾病的關係 1
1.2 生物銀行 2
1.3 慢性腎臟病及透析造成之搔癢 3
1.4 痤瘡相關簡介 6
1.5 痤瘡丙酸桿菌相關背景介紹 7
1.6 人類菌相分析 8
第貳章、研究目標 9
第参章、實驗方法 10
3.1 人類皮膚菌種的分離與鑑定 10
3.2 C9亞油酸異構酶基因的選殖與表現 10
3.3 C9亞油酸異構酶純化 11
3.4 聚丙烯醯胺膠體電泳及西方點墨法 12
3.5 免疫色層快篩試紙原理及構造(見圖1) 12
3.6 免疫色層快篩試紙製作流程 13
第肆章、實驗結果 14
4.1 皮膚益生微生物菌組銀行資訊 14
4.2 分離自人類皮膚培養於 Pikovskaya′s Agar上的菌落 14
4.3 C9亞油酸異構酶基因的擴增 14
4.4 IPTG誘導大腸桿菌BL21大量表現 C9亞油酸異構酶基因 14
4.5 純化轉殖成功之大腸桿菌 BL21之聚丙烯醯胺膠體電泳 14
4.6 純化轉殖成功之大腸桿菌 BL21之西方點墨法 15
4.7 人類微生物總體之加熱破膜菌液可應用於免疫色層快篩試紙 15
4.8 人類微生物總體純化之蛋白可應用於人類免疫色層快篩試紙 15
第伍章、討論 28
參考文獻 31
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指導教授 黃俊銘(Chun-Ming Huang) 審核日期 2016-8-26
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