微生物群對人類健康的有益影響：小鼠研究對解澱粉芽孢桿菌和 痤瘡丙酸桿菌脂肪酶的新見解

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薩恩貝(Enkhbat Zayabaatar) 查詢紙本館藏

畢業系所

生醫科學與工程學系

論文名稱

微生物群對人類健康的有益影響：小鼠研究對解澱粉芽孢桿菌和痤瘡丙酸桿菌脂肪酶的新見解
(Beneficial effects of microbiota in human health: Novel Insights from Mice Studies on Bacillus amyloliquefaciens and Cutibacterium acnes Lipase)

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至系統瀏覽論文 (2028-7-5以後開放)

摘要(中)

微生物群能夠通過調節免疫系統、代謝營養物質和抵禦包括細菌和病毒在內的病原體，
在維持人類健康方面發揮著至關重要的作用。微生物群失調或失衡與多種疾病有關，
表明了微生物群與人類健康之間相互作用的重要性。在本研究中，我們首先研究液化
澱粉芽孢桿菌(Bacillus amyloliquefacien, B. amyloliquefaciens)對發芽水稻種子 γ-胺基丁酸
(Gamma-aminobutyric acid, GABA) 積累的影響，從而增加小鼠背部皮膚中的 I 型膠原
蛋白 (type I collagen, COL1)。不管有沒有液化澱粉芽孢桿菌對發芽水稻進行孵育，都能
從中檢測到 GABA 含量，並且使用蛋白質轉漬法能夠檢測到大量的 COL1。結果表明，
局部應用與液化澱粉芽孢桿菌共培養水稻種子的上清液可以顯著增加小鼠皮膚中
COL1 的產生。其次，我們研究了痤瘡丙酸桿菌(Cutibacterium acnes, C. acnes)的脂肪酶
或棕櫚酸是否可以促進小鼠產生針對 SARS-CoV-2 膜蛋白（ membrane glycoprotein M）
的抗體。每隔 2 周使用膜蛋白加上脂肪酶或棕櫚酸對小鼠進行 3 次皮下免疫注射。並通
過酵素連結免疫吸附分析法(Enzyme Linked Immune Sorbent Assay, ELISA) 對小鼠肺部
中的白細胞介素-6(level interleukin-6, IL-6)水平進行定量。我們的數據表明，與小鼠單獨
使用膜蛋白的免疫相比，使用膜蛋白加脂肪酶或棕櫚酸的聯合免疫有效誘導了高抗體反
應。此外，在使用膜蛋白加脂肪酶或棕櫚酸免疫的小鼠肺中，由 SARS-CoV-2 膜蛋白誘
導產生的 IL-6 顯著減少。總而言之，這些研究提供了對天然來源的佐劑在 COVID-19
疫苗開發中的潛力以及微生物群中細菌作為治療人類疾病的生物療法的重要性的見解。

摘要(英)

The microbiome has been shown to play a crucial role in maintaining human health by
regulating the immune system, metabolizing nutrients, and protecting against pathogens
including bacteria and viruses. Dysbiosis or imbalance in the microbiome has been linked to
various diseases, highlighting the importance of understanding the interaction between
microbiome and human health. First, we investigated the effect of Bacillus amyloliquefaciens
(B. amyloliquefaciens) on the accumulation of gamma-aminobutyric acid (GABA) in
germinated rice seed. Afterward, we tested the effect of those germinated rice seed on
production of type I collagen (COL1) in the dorsal skin of mice. GABA content were detected
in the germinated rice incubated with or without B. amyloliquefaciens by the colorimetric test.
Further, amount of COL1 were detected using western blot method. The results showed that
the topical application of supernatant from rice seed co-cultivated with B. amyloliquefaciens
can significantly increase the production of COL1 in the dorsal skin of mice. Secondly, we
investigated whether Cutibacterium acnes (C. acnes) lipase or palmitic acid could boost the
antibody production against SARS-CoV-2 membrane glycoprotein (M protein) in mice. Mice
were subcutaneously immunized with M protein plus lipase or palmitic acid 3 times in 2-week
intervals. The level interleukin (IL)-6 in the lung of mice were quantified by enzyme linked
immune sorbent assay (ELISA). Our data demonstrated that co-immunization with M protein
plus lipase or palmitic acid effectively induced a high antibody response in comparison to
immunization with M protein alone in mice. Furthermore, the production of IL-6 induced by
SARS-CoV-2 M protein was drastically reduced in the lung of mice immunized with M protein
plus lipase or palmitic acid. In summary, these studies provide insights into the potential of
naturally derived adjuvants for COVID-19 vaccine development and the importance of bacteria
in the microbiome as biotherapeutics for treatments of human diseases.

關鍵字(中)

★ 痤瘡丙酸桿菌
★ COVID-19 疫苗
★ GABA
★ 成纖維細胞
★ IL-6
★ IAA
★ 脂肪酶
★ M 蛋白
★ GABA 積累
★ 小鼠背部皮膚
★ 棕櫚酸
★ siRNA

關鍵字(英)

★ C. acnes
★ COVID-19 vaccine
★ GABA
★ fibroblast cells
★ IL-6
★ IAA
★ lipase
★ M protein
★ accumulation of GABA
★ dorsal skin of mice
★ palmitic acid
★ siRNA

論文目次

Table of Contents
Abstract in Chinese I
Abstract in English II
Acknowledgments III
Table of Contents V
List of Figures IX
List of Tables. IX
Abbreviations X
Chapter I. Introduction 1
1.1. Microbiome. 1
1.1.1. Human Microbiome 1
1.1.2. Plant microbiome 4
1.3. Human skin and its microbiome 6
1.3.1. Most abundant skin bacteria 6
1.3.2. C. acnes lipase 7
1.3.3. Aging Skin and Type I collagen (COL1) 8
1.4. Plant growth-promoting bacteria 9
1.4.1. B. amyloliquefaciens 9
1.5. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 11
1.5.1 SARS-CoV-2 M protein 12
1.5.2. Interleukin (IL)- 6 and cytokine storm in COVID-19 13
Chapter II. Co-Immunization of Mice with M Protein and Cutibacterium acnes Lipase Enhances the Antibody Responses to Sars-Cov-2 Membrane Glycoprotein and Mitigates the Interleukin-6 Production in Mice Lung 15
2.1. Introduction 15
2.2. Materials and methods 17
2.2.1. Animals 17
2.2.2. Cloning and expression of SARS-CoV-2 M protein and C. acnes lipase 17
2.2.3. Immunization of mice and detection of antibody titer 18
2.2.4. Western blotting 18
2.2.4. Cell culture 19
2.2.5. Gas chromatography-mass spectrometry (GC-MS) 19
2.2.6. Immunohistochemical staining (IHC) 20
2.2.7. Nasal inoculation of recombinant M protein and IL-6 determination 20
2.2.8. Statistical analysis 20
2.3. Results 21
2.3.1. Co-immunization with M protein plus lipase induced high humoral response 21
2.3.2. Co-immunization with M protein plus lipase induced the activation of DCs 22
2.3.3. GC-MS Analysis for the identification of FFAs produced by SZ95 cells 23
2.3.4. Co-immunization with M protein plus palmitic acid induced higher humoral response 26
2.3.5. Recombinant M protein-induced IL-6 in the BALF of mice and in vitro neutralization 27
2.3.6 M protein-induced IL-6 production in BALF of mice immunized with GFP, lipase or M protein. 28
2.4. Discussion 30
2.5 Summary of Chapter II. 32
Chapter III. Bacillus amyloliquefaciens Increases the Gaba in Rice Seed for Upregulation of Type I Collagen in The Skin 33
3.1 Introduction 33
3.2. Materials and methods 34
3.2.1 Animals and housing 34
3.2.2. Bacterial culture 35
3.2.3. Rice cultivation with bacteria and sampling 35
3.2.4. Gas chromatography-mass spectrometry (GC–MS) analysis 35
3.2.5. Quantification of GABA content in rice grain 36
3.2.6. Cell culture 36
3.2.7. siRNA-mediated gene silencing of GABRB3 gene/GABA-A receptor 37
3.2.8. Western blotting 37
3.2.9 Cell viability assay 38
3.2.10. Statistical analysis 38
3.2.11. Identification of bacteria 38
3.3. Results 39
3.3.1. GABA and IAA production in rice co-cultivated with B. amyloliquefaciens EH-9 39
3.3.2. COL1 production in NIH/3T3 with and without GABA 40
3.3.3. COL1 production in the dorsal skin of mice 42
3.3.4. COL1 production in the GABRB3 gene knocked down mice 44
3.3.5. Cell viability assay in NIH/3T3 with GABA 45
3.4. Discussion 46
3.5. Summary of Chapter III. 49
Chapter IV. Future Work 50
Changes In Electricity Generation of Gut Probiotic Lactobacillus Rhamnosus Via Serial Passage 50
4.1. Introduction 50
4.2. Materials and Methods 52
4.2.1. Bacterial culture and fermentation 52
4.2.2. Serial passage 52
4.2.3. Detection of bacterial electricity generation in microbial fuel cells (MFCs) 53
4.2.4. Detection of FMN production 53
4.2.5. Statistical analysis 54
4.3. Results 54
4.3.1. Linoleic acid as a prebiotic to induce fermentation of L. rhamnosus EH8 54
4.3.2. Fermentation ability of L. rhamnosus EH8 under anaerobic/aerobic condition. 55
4.3.3. Electricity production of L. rhamnosus EH8 in MFCs 56
4.3.4. Electricity generation in serial passages of L. rhamnosus EH-8. 58
4.4. Discussion 60
4.5. Limitations in future work. 62
Chapter V. Conclusion and Outlook 63
PUBLICATIONS 64
BIBLIOGRAPHY 66

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指導教授

陳純娟(Chun-Chuan Chen)

審核日期

2023-7-5

推文