博碩士論文 107827028 詳細資訊




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姓名 楊仁和(Jen-Ho Yang)  查詢紙本館藏   畢業系所 生物醫學工程研究所
論文名稱 運用老鼠鼻腔內定植人類表皮葡萄球菌之策略以降低SARS-CoV-2之核殼蛋白於肺中誘導的介白素6
(The strategy of using mice nasally colonized human Staphylococcus epidermidis to minimize the levels of lung interleukin-6 induced by SARS-CoV-2 nucleocapsid phosphoprotein)
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摘要(中) 據報導,嚴重急性呼吸綜合症候群冠狀病毒2 (SARS-CoV-2) 的感染可引發過度的IL-6信號傳導,包括有助於2019年重症冠狀病毒疫情 (COVID-19) 多重器官衰竭的細胞因子風暴。使用人源化的小鼠模型,我們證明鼻腔接種SARS-CoV-2的核帽磷蛋白 (NPP)增加支氣管肺泡灌洗液 (BALF) 中IL-6含量。對表皮葡萄球菌 (S. epidermidis) 定植的小鼠鼻腔施用液態辛酸葵酸椰油酯 (LCC) 可以顯著減輕NPP誘導的IL-6。此外,表皮葡萄球菌介導LCC發酵產生電力和丁酸,分別促進細胞定植和激活游離脂肪酸受體2 (Ffar2)。抑制Ffar2阻礙了表皮葡萄球菌加入LCC對減少NPP誘導IL-6的降低作用。綜上所述,本研究結果表明,鼻腔表皮葡萄球菌可能是緩解SARS-CoV-2呼吸道感染所引起的細胞因子風暴的第一道防線的一部分。

本研究是與本實驗室的博士生高銘杉學長共同完成的。本人的貢獻,包括純化蛋白質、病毒接種、小鼠實驗、IL-6檢測、細菌篩選、肺液採集、電壓測量、DNA純化、PCR反應、細菌發酵以及HPLC實驗等,在本論文中均有介紹。作為共同作者,題為「Nasal colonization of Staphylococcus epidermidis mitigates SARS-CoV-2 nucleocapsid phosphoprotein-induced interleukin-6 in the lung」的手稿標題和摘要作為附錄。
摘要(英) It has been reported that infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) triggered excessive interleukin (IL)-6 signaling, leading to a myriad of biological effects including a cytokine storm that contributed to multiple organ failure in coronavirus disease 2019 (COVID-19). Using a humanized mouse model, we have demonstrated that nasal inoculation of nucleocapsid phosphoprotein (NPP) of SARS-CoV-2 increased IL-6 content in bronchoalveolar lavage fluid (BALF). Nasal administration of liquid coco-caprylate/caprate (LCC) onto Staphylococcus epidermidis (S. epidermidis)-colonized mice significantly diminished NPP-induced IL-6. Furthermore, S. epidermidis- mediated LCC fermentation to generate electricity and butyric acid that promoted bacterial colonization and activated free fatty acid receptor 2 (Ffar2), respectively. Inhibition of Ffar2 suppressed the effect of S. epidermidis plus LCC on the reduction of NPP-induced IL-6. In summary, results in this study suggest that nasal S. epidermidis may be part of the first line of defense in ameliorating a cytokine storm induced by airway infection of SARS-CoV-2.

This study has been conducted with Mr. Ming Shan Kao, a PhD student in our laboratory. My contributions including protein purification, virus inoculation, mouse experiments, Interleukin (IL)-6 assays, bacterial screening, lung fluid collection, voltage measurement, DNA purification, PCR reaction, bacterial fermentation and HPLC experiments, have been described in this thesis. As a co-author, the title and abstract of a manuscript entitled” Nasal colonization of Staphylococcus epidermidis mitigates SARS-CoV-2 nucleocapsid phosphoprotein-induced interleukin-6 in the lung” have been attached as an appendix.
關鍵字(中) ★ 表皮葡萄球菌
★ 新冠病毒
★ 核殼蛋白
★ 介白素6
關鍵字(英) ★ Staphylococcus epidermidis
★ SARS-CoV-2
★ nucleocapsid phosphoprotein
★ interleukin-6
論文目次 目錄

中文摘要 i
Abstract ii
致謝 iii
目錄 iv
圖目錄 vii
表目錄 viii
第壹章、緒論 1
1. 研究背景 1
2. 冠狀病毒及致病 1
2.1 介紹 1
2.2 冠狀病毒之核帽蛋白 (NPP) 2
2.3 細胞因子風暴及介白素6 (IL-6) 2
3. 表皮葡萄球菌 (S. epidermidis) 3
3.1 防禦病毒能力 3
3.2 益生菌活性 3
4. 研究動機 4
第貳章、研究目的及架構 5
第參章、材料與方法 6
1. 使用儀器 6
2. 使用藥品 6
3. 實驗方法 8
3.1 重組NPP的克隆、表達與純化 8
3.1.1 重組NPP來源 8
3.1.2 重組NPP之克隆 8
3.1.3 重組NPP之表達 8
3.1.4 重組NPP之純化 9
3.1.5 十二烷基硫酸鈉聚丙烯醯胺凝膠電泳 9
3.2 鼻腔接種和IL-6檢測 10
3.2.1 重組NPP之鼻腔接種 10
3.2.2 IL-6檢測 10
3.3 表皮葡萄球菌在小鼠鼻腔的定植 11
3.3.1 表皮葡萄球菌之篩選 11
3.3.2 表皮葡萄球菌之培養 11
3.3.3 表皮葡萄球菌在小鼠鼻腔的定植 11
3.3.4 菌種之保存 12
3.3.5 抽取菌種DNA 12
3.3.6 DNA瓊脂醣膠體電泳 12
3.4 發酵實驗 14
3.4.1 pH meter 14
3.4.2 Phenol Red 酸鹼指示劑 14
3.4.3 菌液培養 14
3.5 細菌電量檢測 15
3.5.1 電量檢測 15
3.6 鼻中隔分離與丁酸檢測 15
3.6.1 鼻中隔分離 15
3.6.2 丁酸檢測 15
4. High Performance Liquid Chromatography (HPLC) 16
5. 將NPP接種於已被表皮葡萄球菌定植的小鼠和抑制Ffar2 16
第肆章、結果 18
1. 重組NPP之接種 18
2. 菌種的篩選及鑑定 18
3. 人類鼻腔菌種的篩選及鑑定 19
4. 人類鼻腔發酵LCC和產生電量 19
5. LCC發酵介導發電對表皮葡萄球菌的鼻腔定植至關重要 19
6. 鼻中隔接種表皮葡萄球菌在有LCC的條件下產生丁酸 20
7. 表皮葡萄球菌加LCC通過激活Ffar2減弱NPP誘導的BALF中IL-6的產生 20
第伍章、討論 22
第陸章、結論 25
參考文獻 26
附錄 42
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指導教授 黃俊銘(Chun-Ming Huang) 審核日期 2021-1-26
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