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DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 李財坤 | |
dc.contributor.author | Wei-Hsiang Liao | en |
dc.contributor.author | 廖偉翔 | zh_TW |
dc.date.accessioned | 2021-07-11T15:45:13Z | - |
dc.date.available | 2021-10-11 | |
dc.date.copyright | 2018-10-11 | |
dc.date.issued | 2018 | |
dc.date.submitted | 2018-08-08 | |
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Toxins, 2018. 10(4): p. 151. 13. Secher, T., et al., Escherichia coli producing colibactin triggers premature and transmissible senescence in mammalian cells. PLoS One, 2013. 8(10): p. e77157. 14. Haggar, F.A. and R.P. Boushey, Colorectal cancer epidemiology: incidence, mortality, survival, and risk factors. Clinics in colon and rectal surgery, 2009. 22(4): p. 191. 15. Dejea, C.M., et al., Patients with familial adenomatous polyposis harbor colonic biofilms containing tumorigenic bacteria. Science, 2018. 359(6375): p. 592-597. 16. Boleij, A., et al., Clinical Importance of Streptococcus gallolyticus infection among colorectal cancer patients: systematic review and meta-analysis. Clinical Infectious Diseases, 2011. 53(9): p. 870-878. 17. Yang, Y., et al., Colon macrophages polarized by commensal bacteria cause colitis and cancer through the bystander effect. Translational oncology, 2013. 6(5): p. 596-IN8. 18. Fearon, E.R. and B. Vogelstein, A genetic model for colorectal tumorigenesis. Cell, 1990. 61(5): p. 759-767. 19. Kinzler, K.W. and B. Vogelstein, Lessons from hereditary colorectal cancer. Cell, 1996. 87(2): p. 159-170. 20. Smith, G., et al., Mutations in APC, Kirsten-ras, and p53—alternative genetic pathways to colorectal cancer. Proceedings of the National Academy of Sciences, 2002. 99(14): p. 9433-9438. 21. Levine, A.J., p53, the cellular gatekeeper for growth and division. Cell, 1997. 88(3): p. 323-331. 22. Russo, A., et al., The TP53 colorectal cancer international collaborative study on the prognostic and predictive significance of p53 mutation: influence of tumor site, type of mutation, and adjuvant treatment. ONCOLOGY, 2005. 23: p. 7518-7528. 23. Iacopetta, B., et al., Functional categories of TP53 mutation in colorectal cancer: results of an International Collaborative Study. Annals of oncology, 2006. 17(5): p. 842-847. 24. Iacopetta, B., TP53 mutation in colorectal cancer. Human mutation, 2003. 21(3): p. 271-276. 25. Gatalica, Z., et al., High microsatellite instability (MSI-H) colorectal carcinoma: a brief review of predictive biomarkers in the era of personalized medicine. Familial cancer, 2016. 15(3): p. 405-412. 26. Hewish, M., et al., Mismatch repair deficient colorectal cancer in the era of personalized treatment. Nature reviews Clinical oncology, 2010. 7(4): p. 197. 27. Cuevas-Ramos, G., et al., Escherichia coli induces DNA damage in vivo and triggers genomic instability in mammalian cells. Proc Natl Acad Sci U S A, 2010. 107(25): p. 11537-42. 28. Iranpour, D., et al., Phylogenetic groups of Escherichia coli strains from patients with urinary tract infection in Iran based on the new Clermont phylotyping method. BioMed research international, 2015. 2015. 29. Raisch, J., et al., Colon cancer-associated B2 Escherichia coli colonize gut mucosa and promote cell proliferation. World Journal of Gastroenterology: WJG, 2014. 20(21): p. 6560. 30. Lengauer, C., K. Kinzler, and B. Vogelstein, Genetic instabilities in human cancers. Nature, 1998. 396(6712): p. 643-649. 31. Li, T.-K., et al., DNA topoisomerase III alpha regulates p53-mediated tumor suppression. Clinical Cancer Research, 2014: p. clincanres. 1997.2013. 32. Imesch, P., et al., MLH1-deficient HCT116 colon tumor cells exhibit resistance to the cytostatic and cytotoxic effect of the poly (A) polymerase inhibitor cordycepin (3'-deoxyadenosine) in vitro. Oncology letters, 2012. 3(2): p. 441-444. 33. de las Alas, M.M., et al., Loss of DNA mismatch repair: effects on the rate of mutation to drug resistance. Journal of the National Cancer Institute, 1997. 89(20): p. 1537-1541. 34. Li, X.-L., et al., P53 mutations in colorectal cancer-molecular pathogenesis and pharmacological reactivation. World Journal of Gastroenterology: WJG, 2015. 21(1): p. 84. 35. Ahmed, D., et al., Epigenetic and genetic features of 24 colon cancer cell lines. Oncogenesis, 2013. 2: p. e71. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/79116 | - |
dc.description.abstract | 大腸桿菌是微生物感染中最常見來源之一,同時也是人體腸內菌的一員。近來已發現某些大腸桿菌大有一大段基因名為pks+-island,帶有此基因的大腸桿菌稱為E. coli pks+。目前已知,E. coli pks+會造成細胞DNA斷裂,且細胞若存活下來會產生基因不穩定(genome instability)及癌化現象 (tumorigenesis)。而動物實驗也發現E. coli pks+加上腸道發炎的微環境會形成腫瘤。臨床上也發現大腸癌病患腸道中帶有E. coli pks+的比例較正常人高出2至3倍。大腸癌的發生是藉由一系列的基因突變而造成的,其中已知參與的基因有APS、K-Ras及p53。其中p53的突變是屬於最終一步的突變,並且會使腺癌(adenoma)轉變成惡性的癌瘤 (carcinoma),由此可知p53的正常或突變對於大腸癌化扮演很重要的角色。錯配修復 (mismatch repair)的功能對於大腸癌進程也有相當的影響。研究顯示,部分大腸癌患者的細胞帶有錯配修復的缺失,且也發現錯配修復的缺失的大腸癌患者其腫瘤普遍具有侵略性且更為惡化。目前推論,若癌細胞帶有錯配修復的缺失,細胞易產生二次突變(secondary mutation),倘若突變發生在抑癌基因(tumor suppressor gene)或原致癌基因(proto-oncogene),則會使癌細胞更惡劣。在本篇研究,我們利用多株大腸癌細胞株配合著E. coli pks+以多個面向探討,包括: E. coli pks+造成的DNA 斷裂、E. coli pks+引發的抑制細胞生長、細胞受到E. coli pks+感染後DNA 修復的情形及E. coli pks+引發的癌化現象來探討。此外,我們利用HCT116 和HCT116 p53-/-細胞執行上述的實驗,來探討p53扮演的角色。另外,為了研究錯配修復機制,我們利用HCT116 + chr2 (錯配修復缺失的細胞)及HCT116 + chr3 (錯配修復正常的細胞) 執行上述的實驗,來探討錯配修復扮演的角色。目前實驗結果顯示,在HCT116 和HCT116 p53-/-細胞中,E. coli pks+抑制生長的能力及腫瘤生成能力皆差不多,我們推論p53可能沒有參與E. coli pks+引發的生物反應; 在HCT116 + chr2 及HCT116 + chr3細胞中,我們發現在彗星(comet)實驗中,細胞受到E. coli pks+感染後相較於HCT116 + chr3細胞,HCT116 + chr2產生較多的DNA斷裂,且產生的尾距 (tail moment)也較多。 而我們也發現E. coli pks+對於HCT116 + chr2產生的腫瘤生成能力比HCT116 + chr3還明顯。綜上所述,我們認為錯配修復對於E. coli pks+ 所引發的腫瘤生成能力扮演一定的角色,而在未來或許可以多加探討。 | zh_TW |
dc.description.abstract | Escherichia coli is the most common cause of infections and a commensal of normal gut microbiota. In recent years, Scientists found that some E. coli carry a conserved genomic island named “ pks+ island”. This gene clusters allow for producing a putative genotoxin, Colibactin. According to previous researches, a short exposure of cultured mammalian epithelial cells to live E. coli pks+ would induce a transient DNA damage response, following by cell division with incomplete DNA repair. The exposed cells present a significant increase in anchorage-independent soft agar colony formation, indicating that the infection of E. coli pks+ may cuase genomic instability and mutagenic potential, thus favoring tumor progression. In my project, we try to elucidate the molecular pathways associated with the E. coli pks+ infected colon cells. p53 is a tumor suppressor gene, and plays an important role in colon cancer progression. In colon cacner progression, loss of p53 can be seen as the last step from adenoma to carcinoma. Recently, mismatch repair status is also key factor in cancer development. In colorectal cancer, loss of mismatch repair factor may induce secondary mutation. If secondary mutation is tumor suprressor gene or proto-oncogene, cancer may develop more tumorigenesis and invasion. In this thesis, we first build up the in vitro infection assay protocol. Next, we try to investigate the influence of E. coli pks+ on colon cancer cells from different aspect: Initial damage, recovery time, cell viability and tumorigenesis. We found that infection of E. coli pks+ increases the protein level of γH2AX and activation of phosphorylated ATM and Chk2, which indicated that E. coli pks+ may cause G2/M phase cell cycle arrest. In the other aspect, we attempt to repeat the results of anchorage-independent soft agar colony formation. According to our data, we found that E. coli pks+ would suppress cell proliferation in HCT116, HCT116 p53-/-, LoVo, SW480 and HT29 cell line. E. coli pks+ increases colony number in HCT116, HCT116 p53-/- and LoVo cell line but not in SW480 and HT29 cell line in anchorage-independent soft agar assay. To investigate more, we look back the genetic and mutation status of these colon cancer cell line. We found that mismatch repair (MMR) status may have some correlation with E. coli pks+ –increasing colony number. To confirm this hypothesis, we use HCT116 + chr2 (MMR-) and HCT116 + chr3 (MMR+) cell line as model, and perform the experiments described in previous. Western blot was used to check the MMR status and it showed that HCT116 + chr3 cells expressed MLH1 but HCT116 + chr2 cells did not express. In comet assay, we found that E. coli pks+ induced higher damage rate in HCT116 + chr2 cells (MMR-) compare to HCT116 + chr3 cells (MMR+) when cells were recovered 20 and 44 hours. Finally, combining with colony formation and soft agar assay, pks++E. coli may induce more colonies in HCT116 + chr2 cells and less colonies in HCT116 + chr3 cells. Together, mismatch repair may be involved in E. coli pks+-induced tumorigenicity and it shall be further investigated. | en |
dc.description.provenance | Made available in DSpace on 2021-07-11T15:45:13Z (GMT). No. of bitstreams: 1 ntu-107-R05445108-1.pdf: 2263961 bytes, checksum: 713c2998976f0e99f070926b4bdae1a3 (MD5) Previous issue date: 2018 | en |
dc.description.tableofcontents | 口試委員會審訂書........................................................................................................i
ACKNOWLEDGEMENTS...........................................................................................ii 中文摘要......................................................................................................................iii ABSTRACT.................................................................................................................v CONTENTS...............................................................................................................vii INTRODUCTION..........................................................................................................1 1. E. coli pks+...............................................................................................................1 1.1 Microbiota............................................................................................................1 1.2 Distribution...........................................................................................................1 1.3 Genome and metabolite.......................................................................................2 1.4 E. coli pks+-induced tumorigenicity.....................................................................3 2. Colorectal cancer (CRC)........................................................................................3 2.1 Epidemiology........................................................................................................3 2.2 Risk factors...........................................................................................................4 2.3 Genetic basis of CRC development.....................................................................4 2.4 The role of p53 in CRC........................................................................................5 2.5 Microsatellite instability and mismatch repair status in CRC..............................6 SPECIFIC AIM..............................................................................................................8 MATHERIALS AND METHODS.................................................................................9 Bacteria strains ..........................................................................................................9 Polymerase chain reaction (PCR) ..............................................................................9 Bacterial growth curve.............................................................................................10 Cell lines..................................................................................................................10 In vitro infection assay..............................................................................................11 Western blot analysis................................................................................................11 Alkaline single cell gel-electrophoresis (Comet) Assay...........................................12 Clonogenic Assay.....................................................................................................13 Anchorage-Independent Soft Agar Assay.................................................................13 RESULTS.....................................................................................................................15 1. Prevalence of pks+ island in E. coli (E. coli pks+) in colorectal cancer patient.....15 2. To validate the presence of functional E. coli pks+................................................15 2.1. E. coli pks+ harbor genotoxic gene.................................................................15 2.2. E. coli pks+ harbor genotoxic function..........................................................16 3. To set up the experimental model for identifying the factors involved in the mutagenic and tumorigenic propensities induced by E. coli pks+..........................17 3.1. Cell culture system; colon cancer cell lines...................................................17 3.2. Setting up the best bacterial condition for in vitro infection.........................17 3.3. Signaling pathways activated by E. coli pks+ infection..............................18 4. Investigation of p53 involvement in E. coli pks+-induced tumorigenesis..............19 4.1. The role of p53 in E. coli pks+-induced cell viability..............................19 4.2. p53 involvement in E. coli pks+-induced tumorigenic activity......................19 5. The effects on the tumorigenecity of different colon cancer cell lines exposed to E. coli pks+.............................................................................................................20 5.1. 4 colon cancer cell lines genetic status and E. coli pks+-induced cytotoxicity. .....................................................................................................20 5.2. The cell viability of different colon cancer cell line exposed to E. coli pks+......................................................................................................21 5.3. The soft agar assay of different colon cancer cell line exposed to E. coli pks+......................................................................................................21 6. Investigation of mismatch repair involvement in E. coli pks+-induced tumorigenesis........................................................................................................22 6.1. Confirm the characteristics of mismatch repair proficient cell line.............22 6.2. The role of mismatch repair in E. coli pks+-induced DNA break.............23 6.3. The role of mismatch repair in DNA repair after E. coli pks+ infection............................................................................................................23 6.4. The role of mismatch repair in E. coli pks+-induced cell viability.........24 6.5. Mismatch repair involvement in E. coli pks+-induced tumorigenic activity…..........................................................................................................24 DISCUSSION..............................................................................................................26 TABLE AND FIGURE................................................................................................31 Table 1: the prevalence of B2 strain E. coli and E. coli pks+ in colorectal cancer............................................................................................................................31 Table 2.1: Colorectal cancer cell lines that were used in this thesis............................32 Table2.2 Mismatch repair (MMR) related protein status of colorectal cancer cell lines. ............................................................................................................................32 Table2.3 Genetic background of colorectal cancer cell line .......................................33 Figure 1. E. coli pks+ harbor pks-island which can be detected by PCR.....................34 Figure 2. Bacterial growth curve of E. coli pks- and E. coli pks+................................36 Figure 3. E. coli pks+ induces the expression level of γH2AX in HCT116..............37 Figure 4. E. coli pks+ induces DNA break and increase the level ofγH2AX thus activates T68 Chk2. .....................................................................................................38 Figure 5. Plating efficiency of HCT116 and HCT116 p53-/- exposed to E. coli pks- or E. coli pks+ ..................................................................................................................40 Figure 6. Anchorage-independent growth of HCT116 and HCT116 p53-/- exposed to E. coli pks- or E. coli pks+............................................................................................43 Figure 7. Plating efficiency of LoVo, SW480 and HT29 exposed to E. coli pks- or E. coli pks+........................................................................................................................45 Figure 8 Anchorage-independent growth of LoVo, SW480 and HT29 exposed to E. coli pks- or E. coli pks+...............................................................................................48 Figure 9. The expression level of MLH1 in HCT116 + chr2 and HCT116 + chr3 cell line................................................................................................................................49 Figure 10. E. coli pks+ did not induce chromosomal DNA break in both HCT116 + chr2 and HCT116 + chr3 cell line exposed bacteria after 4 hours. ............................51 Figure 11. E. coli pks+ induces chromosomal DNA break in both HCT116 + chr2 and HCT116 + chr3 cell line exposed bacteria after 20 hours. ..........................................54 Figure 12. E. coli pks+ induces chromosomal DNA break in both HCT116 + chr2 and HCT116 + chr3 cell line exposed bacteria after 44 hours. ..........................................57 Figure 13. Plating efficiency of HCT116 + chr2 and HCT116 + chr3 exposed to E. coli pks- or E. coli pks+.................................................................................................59 Figure 14. Anchorage-independent growth of HCT116 + chr2 and HCT116 + chr3 exposed to E. coli pks- or E. coli pks+..........................................................................63 REFERENCE...............................................................................................................65 | |
dc.language.iso | en | |
dc.title | 探討Mismatch repair在細胞受到攜有pks island的大腸桿菌感染後引發的癌化現象中所扮演之角色 | zh_TW |
dc.title | Study on potential roles of mismatch repair in tumor-promoting propensity of cells infected with
E. coli pks+ | en |
dc.type | Thesis | |
dc.date.schoolyear | 106-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 倪衍玄,賴逸儒 | |
dc.subject.keyword | 腸內菌,大腸桿菌,癌化,大腸癌,錯誤配對修飾, | zh_TW |
dc.subject.keyword | microbiota,E. coli,pks island,tumorigenesis,colon cancer,mismatch repair, | en |
dc.relation.page | 67 | |
dc.identifier.doi | 10.6342/NTU201802578 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2018-08-08 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 微生物學研究所 | zh_TW |
顯示於系所單位: | 微生物學科所 |
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