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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 俞松良 | |
| dc.contributor.author | Zzu-Han Yang | en |
| dc.contributor.author | 楊斯涵 | zh_TW |
| dc.date.accessioned | 2021-06-15T06:14:01Z | - |
| dc.date.available | 2020-08-11 | |
| dc.date.copyright | 2010-09-13 | |
| dc.date.issued | 2010 | |
| dc.date.submitted | 2010-08-12 | |
| dc.identifier.citation | Andrews-Polymenis, H. L., A. J. Baumler, et al. (2010). 'Taming the elephant: Salmonella biology, pathogenesis, and prevention.' Infect Immun 78(6): 2356-2369.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/47710 | - |
| dc.description.abstract | 沙門氏菌(Salmonella)是一種革蘭氏陰性桿菌，屬於腸內菌(Enterobacteriaceae)之一屬。主要可造成急性腸炎、菌血症以及傷寒 (Typhoid fever) 、副傷寒(Paratyphoid fever)等疾病。其感染來源可透過食入汙染的食物與水或是接觸受感染的動物，是食物中毒主要原因之一。
微核醣核酸(microRNAs)是大約21-24nt的non-coding RNA。可以在轉錄後層級(post-transcription level)調控基因的表現。近年來有許多研究指出，微核醣核酸在Host-pathogen interaction也就是宿主細胞抵禦微生物感染當中扮演著相當重要的角色。此外，微核醣核酸與宿主細胞的Innate immunity的調控亦有著重要的關聯。 在此研究中，我們採用鼠傷寒沙門氏菌(Salmonella Typhimurium) 與人類腸道上皮細胞株(HT-29)為感染實驗模組，欲探討當HT-29細胞受沙門氏菌感染時，腸道上皮細胞本身微核醣核酸之調控以及表現量的差異，並期望找出對於細菌在細胞中複製或與宿主細胞之存活和凋亡有影響力的微核醣核酸，甚至進一步找出這些重要的微核醣核酸的目標基因。 實驗過程中，為了找尋細胞感染時最適當的表現型 (phenotype)，嘗試了細胞存活檢測（MTT cell viability assay）、乳酸去氫酶分析法 (Lactate dehydrogenase assay；LDH assay).、流式細胞儀 (Flow Cytometer)、免疫螢光染色(Immunofluorescent stain)以及細胞內細菌複製曲線 (intracellular growth)等方法。最後以細菌在細胞內的複製曲線為主要研究的表現型，並收集未感染以及感染後 4、16與24小時 等三個時間點的HT-29細胞RNAs，運用TLDA (TaqMan Low Density Array)分析微核醣核酸表現圖譜。 由TLDA之結果，並以qRT-PCR進行再次確認，挑出了四個感染前後表現有顯著差異的微核醣核酸 (hsa-miR-125b、hsa-miR-545、hsa-miR-210以及hsa-miR-146a)，並建構此四個微核醣核酸的表現質體 (p-silencer)並挑選穩定表現此四個微核醣核酸之HT-29 細胞株。在可大量表現這四個微核醣核酸之HT-29細胞株，進行細菌感染實驗，觀察細胞內細菌複製曲線(intracellular growth)的改變。此外，我們運用網路上的軟體預測出DUSP1 (MKP-1)可能為miR-545之目標基因，並且由基因表現晶片及qRT-PCR之實驗結果發現感染16小時及24小時後的DUSP1 mRNA 表現量，明顯地較未感染及感染後4小時下降許多。因此，我們推測，當鼠傷寒沙門氏菌感染宿主細胞時，沙門氏菌使宿主細胞調控miR-545，使miR-545表現量上升，有可能因此而抑制DUSP1的表現，使得鼠傷寒沙門氏菌得以在細胞內複製。 | zh_TW |
| dc.description.provenance | Made available in DSpace on 2021-06-15T06:14:01Z (GMT). No. of bitstreams: 1 ntu-99-R97424011-1.pdf: 6542490 bytes, checksum: b48a17fb2f1d50c42791cbd30c8291b0 (MD5) Previous issue date: 2010 | en |
| dc.description.tableofcontents | CONTENTS
中文摘要……………………………………………………….……….……….………6 ABSTRACT 8 1. INTRODUCTION 10 1.1 Salmonella spp. 11 1.2 MicroRNAs 14 1.3 MicroRNAs, host cells and pathogens 15 1.4 MiRNAs-based therapies 16 1.5 Objectives in the thesis…………………..………………………….…….17 2. MATERIALS AND METHODS 19 2.1 Cell lines 20 2.2 Bacterial Strains and growth conditions 20 2.3 in vitro Infection Protocol 20 2.4 Intracellular growth of Salmonella Typhimurium in HT-29 cells 21 2.5 Immunofluorescent stain 21 2.6 Cell apoptosis assay 22 2.7 Cell viability assay 22 2.8 Cell proliferation assay 23 2.9 RNA isolation 23 2.10 Microarray analysis of miRNA expression 24 2.11 RNA reverse transcription for miRNAs 24 2.12 Quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) for miRNAs 25 2.13 Computational Targeted Gene Predictions of miR-545 25 2.14 RNA reverse transcription of total RNA 26 2.15 SYBR GREEN Quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) for DUSP1 27 2.16 Gene Expression array 27 2.17 Plasmids constructions for precursor-miRNA125b, 146a, 210, 545 28 2.18 P-MIR Luciferase constructions for DUSP1 3’UTR and DUSP1 3’UTR mutant 28 2.19 Cloning of DUSP1 CDS to pcDNA 3.1 29 2.20 miRNAs delivery to HT-29 cells and stable clone selection 30 2.21 Luciferase assay for miR-545 and DUSP1 3’UTR 30 3. RESULTS 32 3.1 Salmonella Typhimurium is sensitive to Gentamicin 33 3.2 25 M.O.I. is the most appropriate condition for Salmonella infection and intracellular growth in HT-29 cells tested by intracellular CFU 33 3.3 25 M.O.I. is the most appropriate condition for Salmonella infection and intracellular growth in HT-29 cells tested by Immunofluorescence 34 3.4 Time course of cell viability assayed by LDH is not a good phenotype for Salmonella infection to HT-29 cells 34 3.5 Time course of intracellular CFU in HT-29 cells infected with Salmonella Typhimurium 35 3.6 Cell apoptosis of HT-29 cells infected with Salmonella Dublin 36 3.7 Cell proliferation of HT-29 cells is not a good phenotype for Salmonella infection to HT-29 cells………………………………………………...…………36 3.8 Taqman Low Density Array miRNAs profile 37 3.9 miRNAs expression level were validated by qRT-PCR 37 3.10 DUSP1 is a possible target gene for miR-545 38 3.11 The down –regulation of DUSP1 mRNA level was validated by SYBR green qRT-PCR 39 4. DISCUSSION 40 4.1 Phenotype selection of Salmonella infection to HT-29 cells 41 4.2 miR-125b down-regulation and its functions 42 4.3 miR-146a up-regulation and its functions 42 4.4 miR-210 up-regulation and its functions 44 4.5 miR-545 up-regulation and its functions 45 4.6 Dusp1: a possible target gene for miR-545 45 5. FIGURES 47 Fig 1. Gentamicin killing assay of STM14028, STLT2 and DH5a. 48 Fig 2. Infection Rate tested by intracellular growth of Salmonella in HT-29 cells……………………………………………………………………………..… 49 Fig3. Infection Rate of different STM14028 M.O.I. tested by Immunofluorescence……………………………………………………………...50 Fig 4 Infection Rate tested by immunofluorescent staining with 10, 25 and 50 M.O.I……………………………………………………………………………....51 Fig 5. Time course of cell viability assayed by LDH assay during STM14028 infection………………………………………………………………………..…..52 Fig 6. Time course of cell viability assayed by MTT during STM14028 infection……………………………………………………………………............53 Fig 7 . Cell apoptosis of HT-29 with infection of 50 M.O.I Salmonella Dublin 24h post infection………………………………………………………..……....54 Fig 8. Cell apoptosis of HT-29 with infection of 50 M.O.I. Salmonella Dublin 48h post infection……………………………………………………………..…...55 Fig 9. Time course of intracellular CFU in HT-29 infected with STM14028…56 Fig 10. Immunofluorescent staining of Salmonella –infected HT-29 in 4, 16, 24 h post infection and non-infected control cells……………………………….....57 Fig 11. Isolated RNA quality from HT-29 cells in four indicated times…………………………………………………………………………..……58 Fig 12. Taqman Low density array miRNAs profile : Expression of 4 h compared with 0 h with over two folds change ………………………………...59 Fig 13. Taqman Low density array miRNAs profile: Expression of 16 h compared with 0 h with over two folds change ………………………………...60 Fig 14. Taqman Low density array miRNAs profile: Expression of 24 h compared with 0 h with over two folds change…………………………………61 Fig 15. Taqman Low density array miRNAs profile: miRNAs with over two folds change in 4, 16 and 24 h post infection…………………………………….62 Fig 16. Relative Expression of Selective miRNAs in TLDA profile……………………………………………………………….......................63 Fig 17. Relative Expression of Selective miRNAs : qRT-PCR validation………………………….........................................................................64 Fig 18. Relative Expression of DUSP1 mRNA in four indicated times: Expression array………….....................................................................................65 Fig 19. Relative Expression of DUSP1 mRNA in four indicated times: qRT-PCR validation………………................................................................................66 Fig 20. The Binding motif of miR-545 and the 3’UTR of DUSP1( from RNAhybrid)………………….................................................................................67 6.TABLES……….………………………………………………………….….………68 Table 1 : Relative Expression of Selective miRNAs in TLDA profile….….…..69 Table 2: Relative Expression of Selective miRNAs in sample 1: qRT-PCR validation…………………………………………………….………………..…….70 Table 3: Relative Expression of Selective miRNAs in sample 2: qRT-PCR validation……………………………………………………………………...…….71 Table 4: Taqman Low density array miRNAs profile: miRNAs with over two folds change in 4, 16 and 24 h post infection………………………….…..………72 Table 5: SYBR GREEN qRT-PCR primer sequence of DUSP1……….…..…...74 Table 6: Primers for construction of miRNAs………………………….….……75 Table 7 : Primers for construction of DUSP1 3’UTR and 3’UTR mut ..….…..76 Table 8 : miRNAs and their functions ……………………….............……..77 | |
| dc.language.iso | en | |
| dc.title | 微核醣核酸於沙門氏菌感染宿主細胞之角色探討 | zh_TW |
| dc.title | Roles of microRNAs in the interaction between
Salmonella and Host cells | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 98-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 鄧麗珍,廖淑貞,顏伯勳 | |
| dc.subject.keyword | 沙門氏菌,微核醣核酸,細菌複製曲線,DUSP1,腸宿主細胞與病原體之交互作用, | zh_TW |
| dc.subject.keyword | Salmonella,microRNAs,bacteria intracellular replication,DUSP1,host- pathogen interaction, | en |
| dc.relation.page | 83 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2010-08-12 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 醫學檢驗暨生物技術學研究所 | zh_TW |
| 顯示於系所單位： | 醫學檢驗暨生物技術學系 | |
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