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研究生:曾郁雯
研究生(外文):Yu-Wen Tseng
論文名稱:微核醣核酸146a於沙門氏菌感染宿主細胞之角色探討
論文名稱(外文):Role of Host microRNA-146a in Salmonella Infection
指導教授:俞松良俞松良引用關係
口試委員:賴信志楊翠青顏伯勳邱浩傑
口試日期:2014-06-26
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:醫學檢驗暨生物技術學研究所
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2014
畢業學年度:102
語文別:英文
論文頁數:79
中文關鍵詞:沙門氏菌微核醣核酸微核醣核酸146a胞內繁殖病原體-宿主交互作用高通量細胞影像分析系統
外文關鍵詞:SalmonellamiRNAsmiR-146aintracellular replicationpathogen-host interactionhigh content analysis
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沙門氏菌(Salmonella)是一種可以在細胞內生長的革蘭氏陰性菌,沙門氏菌會藉由糞口傳染且可以感染人和動物,是目前造成食物中毒主要的病原菌之一。沙門氏菌會感染吞噬細胞與非吞噬細胞並在宿主細胞內進行複製,當宿主細胞被沙門氏菌感染時,細胞會啟動初級免疫反應以對抗沙門氏菌的感染。已發現微核醣核酸(microRNAs)會參與宿主細胞抵抗病原菌的防禦,目前已有許多研究證實微核醣核酸可以調控初級免疫反應、發炎反應以及抗微生物感染的免疫反應。然而,微核醣核酸在胞內細菌感染中所扮演的角色仍未清楚。在我們的研究當中,我們利用沙門氏菌感染人類大腸上皮細胞HT-29後,發現宿主細胞的miR-146a表現量會增加。為進一步探討宿主miR-146a在沙門氏菌感染時所扮演的角色,我們使用沙門氏菌感染大量表現miR-146a的HT-29細胞株,再利用菌落形成單位(colony formation unit)分析細胞內細菌數以觀察細菌在細胞內生長的情形。結果發現在大量表現miR-146a的HT-29細胞株中的細菌數比對照組有明顯減少。為了更進一步探討miR-146a是否具有抑制細胞內細菌的能力,我們另外選用HCT116和HeLa兩種細胞株,進行沙門氏菌感染並觀察其是否具有相同現象。首先我們利用MTT證實miR-146a在細胞內大量表現後並不會影響細胞的生長速度。接著再利用先前實驗方法感染沙門氏菌並進行分析,從實驗結果中發現當細胞有表現miR-146a時,細胞內的細菌數的確會比對照組少,而在細胞數目上則沒有明顯的差異。此外我們也使用高通量細胞影像分析系統(high content analysis)作為連續觀察細胞內沙門氏菌生長情形的影像工具,以HT-29和HeLa細胞進行觀察,此影像系統分析的結果與菌落形成單位的結果相符合。

Salmonella, a Gram negative intracellular bacterium is an important and widespread pathogen causing foodborne infections in both human beings and animals. Salmonella can infect phagocytic and non-phagocytic cells, surviving and replicating within host cells. The innate immunity of host cells is triggered to against Salmonella infection. Regarding to the host responses against pathogens, it has been reported that microRNAs (miRNAs) are involved in this process, and miRNAs have been identified as an important regulator of innate immunity, inflammatory response and anti-microbial immunity. However, the role of miRNA in host response to intracellular bacterial infection is still largely unclear.
In our study, we found that miR-146a was induced in HT-29 cells after Salmonella infection. To understand the role of miR-146a in Salmonella infection, Salmonella was used to infect miR-146a-overexpressed HT-29 cells, and the colony formation unit (CFU) assay was used to measure the intracellular bacterial numbers after Salmonella infection. The results showed that the intracellular CFU was significantly decreased in miR-146a-transfected cells compared with the mock cells. To determine whether the inhibitory activity of miR-146a is a universal phenomenon, we measured the impact of miR-146a on two other Salmonella-infected epithelial cells, HCT116 and HeLa cells, and found that miR-146a did not affect cell viability of HCT116 and HeLa cells assayed by MTT assays. After Salmonella infection, the results showed that the intracellular CFU was decreased in miR-146a expressed cells compared with the mock cells. Furthermore, we also used a high-throughput microscopy, high content analysis (HCA), as an image tool to investigate the intracellular growth of Salmonella continuously in both HT-29 cells and HeLa cells, demonstrating that the data were consistent with the CFU assay.

口試委員會審定書 I
Acknowledgement II
中文摘要 III
Abstract IV
1. Introduction 1
1.1. Introduction of Salmonella spp. 2
1.1.1. Pathogenesis of Salmonella 2
1.2. Host responses to enteric bacterial infection 4
1.2.1. Epithelial integrity and epithelial cell turnover 5
1.2.2. Innate immune responses 5
1.2.3. Autophagy 6
1.3. MicroRNAs 7
1.3.1. MicroRNAs against pathogen infection 8
1.4. Aim of this study 11
2. Materials and Methods 13
2.1. Bacteria strains and culture conditions 14
2.2. Cell culture and transfection 14
2.3. In vitro infection assay 15
2.4. Enumeration of intracellular bacteria 16
2.5. Cell viability assay 16
2.6. RNA isolation 17
2.7. Reverse transcription for miRNAs 18
2.8. Quantification of miRNAs by qRT-PCR 18
2.9. Construction of miRNAs expressing plasmids 19
2.10. Establish miR-146a mix clone in HCT116 cells and HeLa cells 19
2.11. MTT cell viability assay 20
2.12. Image-based high content assay 21
2.13. Statistical analysis 23
3. Results 24
3.1. The optimal condition for Salmonella infection in HT-29 cells 25
3.2. The impacts of identified host miRNAs on intracellular replication of Salmonella 26
3.3. Establishment mixed stable clone in other epithelial cells 27
3.4. Time course of intracellular CFU in HCT116 and HeLa cells infected with Salmonella 29
3.5. The expression of miR-146a in HCT116 and HeLa cells limits intracellular replication of Salmonella 29
3.6. Imaged-based high content assay for monitoring the intracellular replication of Salmonella 31
4. Discussion 33
5. Figures 42
Figure 1. Optimization of the multiplicity of infection. 44
Figure 2. Overexpression of microRNAs in HT-29 cells. 46
Figure 3. The transient overexpression of host miRNAs may restrict Salmonella intracellular replication 49
Figure 4. The transient overexpression of host miR-146a, miR-182* and miR-376a limits Salmonella intracellular replication 50
Figure 5. Expression of miR-146a in HCT116 and HeLa epithelial cells 53
Figure 6. Time course of intracellular CFU in HT-29 infected with Salmonella. 54
Figure 7. The miR-146a inhibits Salmonella intracellular replication in epithelial HCT116 and HeLa cells. 55
Figure 8. Monitoring in vitro Salmonella infection assay by high content analysis 60
6. References 64



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