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Title: | 微核醣核酸146a於沙門氏菌感染宿主細胞之角色探討 Role of Host microRNA-146a in Salmonella Infection |
Authors: | Yu-Wen Tseng 曾郁雯 |
Advisor: | 俞松良 |
Keyword: | 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, Salmonella,miRNAs,miR-146a,intracellular replication,pathogen-host interaction,high content analysis, |
Publication Year : | 2014 |
Degree: | 碩士 |
Abstract: | 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. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/57573 |
Fulltext Rights: | 有償授權 |
Appears in Collections: | 醫學檢驗暨生物技術學系 |
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