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研究生：	薛方婷 Hsueh, Fang-Ting
論文名稱：	探討口腔普雷沃氏菌對食道鱗狀細胞癌的影響 The influence of Segatella oris on esophageal squamous cell carcinoma
指導教授：	陳振暐 Chen, Jenn-Wei
學位類別：	碩士 Master
系所名稱：	醫學院 - 微生物及免疫學研究所 Department of Microbiology & Immunology
論文出版年：	2025
畢業學年度：	113
語文別：	英文
論文頁數：	69
中文關鍵詞：	口腔普雷沃氏菌、食道鱗狀細胞癌、細胞增生
外文關鍵詞：	Segatella oris, esophageal squamous cell carcinoma, cell proliferation
相關次數：	點閱：66 下載：0
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食道癌在全球十大癌症相關死亡原因中佔有一席之地，是一種進展快速、預後不良的惡性腫瘤。由於缺乏早期臨床症狀，多數患者在確診時已屬晚期，導致整體五年存活率偏低，僅約百分之二十。食道癌主要分為兩種組織型態，其中食道鱗狀細胞癌約佔所有食道癌的九成，好發於食道上三分之二處，常見於亞洲與非洲地區，且男性發生率高於女性。食道鱗狀細胞癌的致病機轉與食道黏膜長期接觸致癌物質有關，例如：菸草中的亞硝胺、酒精代謝產物乙醛。非食道癌患者的食道菌群主要由厚壁菌門、變形菌門與擬桿菌門組成，然而在食道鱗狀細胞癌患者中，厚壁菌門與擬桿菌門的豐度增加，並且常見卟啉單胞菌與普雷沃氏菌等屬。口腔普雷沃氏菌是一種革蘭氏陰性的厭氧桿菌，常見於人類口腔並與多種口腔疾病相關，近年亦有報導指出口腔普雷沃氏菌可引起腦膿瘍、腦膜炎、胸肺感染及菌血症等口腔外感染。根據16S rRNA定序結果，我們發現口腔普雷沃氏菌在食道鱗狀細胞癌患者中顯著上升，顯示其可能具有致病潛力。為了探討這點，我們將口腔普雷沃氏菌與兩株食道鱗狀細胞癌的細胞株共同培養。在CE81T細胞中，與活菌共同培養促進細胞增生，而經過紫外線滅活的口腔普雷沃氏菌則無此效果，揭示促進增生之因子可能來自其分泌物。隨後我們製備口腔普雷沃氏菌的條件培養基，用以處理細胞後亦觀察到明顯增生效果，並且此效果可被加熱或蛋白酶 K消除，代表活性因子可能為蛋白質。以流式細胞儀分析細胞週期，驗證條件培養基造成的細胞增生並非因細胞週期明顯改變所造成的。另一方面，使用兩種細胞遷移實驗證實條件培養基對細胞遷移沒有影響。所有的實驗也在另一株食道鱗狀細胞癌的細胞KYSE-150進行並觀察到類似現象，支持口腔普雷沃氏菌可普遍促進食道鱗狀細胞癌細胞增生之可能。

Esophageal cancer (EC) is among the top ten causes of cancer-related deaths worldwide. It is a rapidly progressive malignant tumor characterized by poor prognosis. Due to the absence of early clinical symptoms, most patients are diagnosed at an advanced stage, resulting in a low overall five-year survival rate of approximately 20%. EC is primarily classified into two major histological types, with esophageal squamous cell carcinoma (ESCC) accounting for about 90% of cases. ESCC typically arises in the upper two-thirds of the esophagus and is more prevalent in Asia and Africa, with a higher incidence in men than in women. The pathogenesis of ESCC involves chronic exposure of the esophageal mucosa to carcinogens, such as nitrosamines in tobacco and acetaldehyde in alcohol. In healthy individuals, the esophageal microbiota is mainly composed of Firmicutes, Proteobacteria, and Bacteroidetes. In contrast, patients with ESCC exhibit increased abundances of Firmicutes and Bacteroidetes, particularly genera such as Porphyromonas and Prevotella. Segatella oris is a Gram-negative anaerobic rod commonly found in the human oral cavity and is frequently associated with oral diseases. Recent case reports have also identified S. oris in extraoral infections, including brain abscess, meningitis, pleuropulmonary infections, and bacteremia. Based on 16S rRNA sequencing analysis, we found that the abundance of S. oris is significantly higher in ESCC patients than in healthy individuals, suggesting a potential pathogenic role. To explore this possibility, we co-cultured S. oris with two ESCC cell lines. In CE81T cells, cell proliferation was significantly enhanced when co-cultured with live S. oris, whereas UV-killed S. oris had no effect, suggesting that secreted factors from S. oris are responsible for promoting proliferation. We then prepared conditioned medium (CM) containing secreted components from S. oris. Treatment of cells with CM similarly promoted proliferation. This effect was abolished by heat inactivation and proteinase K treatment, indicating that the active factor is likely a protein. Flow cytometry analysis demonstrated that the proliferation induced by CM was not mediated through obvious alterations in the cell cycle. Besides, two different migration assays confirmed that CM had no significant effect on cell migration. Similar results were observed in another ESCC cell line, KYSE-150, supporting the generality of S. oris-mediated effects on ESCC cell proliferation.

口試證明 I 
中文摘要 II 
ABSTRACT III 
致謝 V 
CONTENTS VI 
ABBREVIATIONS IX 
Chapter 1. INTRODUCTION 1 
1.1 Esophageal cancer 1 
1.2 The role of cell proliferation and migration in cancer cells 2 
1.3 The microbiota of ESCC patients 3 
1.4 Segatella oris 4 
Chapter 2. MATERIALS AND METHODS 6 
2.1 Bacterial strains and culture conditions 6 
2.2 Cell lines and culture conditions 6 
2.2.1 Esophageal squamous cell carcinoma cells 6 
2.2.2 Oral squamous cell carcinoma cells 7 
2.3 S. oris growth curve 7 
2.3.1 Spectrophotometer 7 
2.3.2 ODBox-C 8 
2.4 S. oris conditioned medium (CM) 8 
2.5 S. oris oxygen tolerance 8 
2.6 Cell proliferation assay 9 
2.6.1 Cell counting 9 
2.6.2 S. oris preparation 9 
2.6.3 Killed S. oris preparation 9 
2.6.4 Co-culture of cells and S. oris 10 
2.6.5 Cell treatment with CM 10 
2.6.6 Cell treatment with pretreated CM 11 
2.6.7 Cell treatment with fractionated CM 11 
2.7 Cell migration assay 12 
2.7.1 Transwell migration assay 12 
2.7.2 Wound healing assay 12 
2.8 Cell cycle 13 
2.9 Statistical analysis 13 
Chapter 3. RESULTS 14 
3.1 S. oris ATCC33573 grow condition 14 
3.2 S. oris ATCC33573 promotes CE81T and KYSE-150 cell proliferation 14 
3.3 CM promotes CE81T and KYSE-150 cell proliferation 15 
3.4 The active component of CM is proteinaceous 16 
3.5 The >100 kDa fraction promotes CE81T and KYSE-150 cell proliferation 17 
3.6 The >100 kDa fraction is trypsin-resistant and sensitive to heat and proteinase K 17 
3.7 The >100 kDa fraction has no obvious effect on CE81T and KYSE-150 cell cycle 18 
3.8 CM has no effect on CE81T and KYSE-150 cell migration 18 
Chapter 4. DISCUSSION 20 
REFERENCES 24 
Figure 1. Growth curve of S. oris ATCC33573 31 
Figure 2. Cell proliferation of CE81T and KYSE-150 cells when co-cultured with S. oris ATCC33573 33 
Figure 3. Cell proliferation of CE81T and KYSE-150 cells when co-cultured with UV-killed S. oris ATCC33573 35 
Figure 4. Cell proliferation of CE81T and KYSE-150 cells when treated with different percentages of CM 37 
Figure 5. Cell proliferation of CE81T cells after using heat-inactivated CM or CM pretreated with various enzymes 39 
Figure 6. Cell proliferation of CE81T and KYSE-150 cells when treated with whole or fractionated CM 41 
Figure 7. Cell proliferation of CE81T and KYSE-150 cells when co-treated with CM (>100 kDa) and various factors 45 
Figure 8. Cell cycle of CE81T and KYSE-150 cells when treated with CM (>100 kDa) 50 
Figure 9. Transwell migration of CE81T and KYSE-150 cells when treated with CM 51 
SUPPLEMENTARY FIGURES 52 
Supplementary Figure 1. Growth curve of S. oris ATCC33573 in VACUETTE® TUBE 52 
Supplementary Figure 2. Oxygen tolerance of S. oris ATCC33573 53 
Supplementary Figure 3. Cell proliferation of CE81T cells after using heat-inactivated CM (>30 kDa) 54 
Supplementary Figure 4. Wound healing of CE81T and KYSE-150 cells when treated with CM 57 
Supplementary Figure 5. Cell proliferation of SCC-15 cells when treated with different percentages of CM 58
                                    

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2026-08-26公開

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