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???org.dspace.app.webui.jsptag.ItemTag.dcfield??? | Value | Language |
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dc.contributor.advisor | 張逸良(Yih-Leong Chang) | |
dc.contributor.author | Yi-Ying Chen | en |
dc.contributor.author | 陳怡穎 | zh_TW |
dc.date.accessioned | 2021-06-17T00:16:23Z | - |
dc.date.available | 2017-09-18 | |
dc.date.copyright | 2012-09-18 | |
dc.date.issued | 2012 | |
dc.date.submitted | 2012-07-03 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/65951 | - |
dc.description.abstract | 鼻咽癌是中國南方、香港、新加坡及台灣等地的華人好發的癌症,其致病原因相當複雜,至今仍尚未被研究清楚。雖然早期Epstein-Barr virus (EBV)被認為和鼻咽癌的發生有很大的關連性,但是近期許多的研究報導都認為EBV並不是導致鼻咽癌發生的原因而是促使鼻咽癌惡化的因素。本論文研究的主要目的,就是想找出與鼻咽癌致病相關的基因。我們使用cDNA微陣列分析(cDNA microarray analysis)的方法,比較鼻咽癌細胞和正常鼻咽黏膜上皮細胞之間的表現量,再經由及時定量聚合酶連鎖反應(Quantitative RT-PCR)及西方墨點法(Western Blotting)發現SRY (sex-determining region Y)-box 5 (SOX-5)基因在鼻咽癌細胞中的表現量有顯著增加。進一步研究SOX5基因表現量增加的分子機制時,我們發現Chemokine (C-C motif) ligand 28 (CCL28) 在鼻咽癌細胞中亦有顯著增加的表現量,在各種不同的鼻咽癌細胞株中,發現CCL28在undifferentiated carcinoma (lymphoepitheliomatous carcinoma of nasopharynx, LE-NPC) NPC-TW03中表現量增加最多。在50個LE-NPC 檢體中,發現(46%)病患之癌細胞有高表現的CCL28。為了進一步確定SOX5和CCL28之間的關係,我們發現SOX5和CCL28基因在鼻咽癌細胞中可相互調控。進而深入研究CCL28對鼻咽癌的病理機理所佔的角色及其功能,我們構建pBIG2i-CCL28質體來轉染鼻咽癌細胞。我們發現轉染的腫瘤細胞可向上調控CCL28的mRNA和蛋白質。CCL28基因在體外培養實驗中,發現到會促進鼻咽腫瘤細胞的生長、移動、侵犯及吸引淋巴球之功能,且以B淋巴球為多。對EBV 感染的B 細胞,CCL28照樣也有吸引EBV+ B細胞之功能。另一方面,我們也發現EBV能感染CCL28高表現的鼻咽癌細胞,並在細胞內複製。然而,帶有CCL28基因表現的鼻咽癌細胞在免疫不全的老鼠中生長時,此基因輕微增加腫瘤生長及轉移能力,卻未有吸引小鼠淋巴球之功能。我們用cDNA microarray觀察到當CCL28基因表現上升後,很多致癌基因表現量上升,另外也有很多抑癌基因表現量下降。由此推論,CCL28基因在鼻咽癌的腫瘤進展中扮演一種類似促使淋巴上皮性鼻咽癌形成的因素並使其生長移動及侵犯性加速。 | zh_TW |
dc.description.abstract | Nasopharyngeal Carcinoma (NPC) is one of the most common cancers among Chinese living in southern China, Hong Kong, Singapore and Taiwan. The cause of the disease is quite complex, and the molecular mechanisms involved in the pathogenesis of NPC still are not yet well defined. Although it was proposed that Epstein-Barr virus (EBV) is closely associated with NPC pathogenesis, but recently many studies have reported that EBV behaves more likely as progression factors but not initiation factors. The purpose of this research was to find out the genes associated with NPC pathogenesis. Using cDNA microarray analysis of mRNA expression between NPC cell lines and normal nasal mucosal epithelial cells, SOX5 gene expression was found significantly increased in NPC cell lines by quantitative RT-PCR and Western blot analysis. In our previous studies of the function of SOX-5 gene in NPC, we found that Chemokine (CC motif) of ligand 28 (CCL28) was also significantly increased in NPC cell lines, especially in NPC-TW03 line which is a lymphoepitheliomatous carcinoma of nasopharynx (LE-NPC). In 50 cases of LE-NPC biopsy specimens we found that about 46% of cases revealed high expression of CCL28 protein in some tumor cells. To further identify the relationship between SOX5 and CCL28, we performed some investigation to clarify this condition. We found that SOX5 and CCL28 gene expressions could be reciprocally regulated in NPC cells and suggested that CCL28 may be a critical factor for the formation of LE-NPC. To study the role of CCL28 in the molecular pathogenesis of LE-NPC and its functions, we constructed a stable pBIG2i-CCL28 transfected NPC cell lines. We found that tumor cells could be up-regulated to express CCL28 mRNA and protein remarkably in those transfectants. This gene could promote tumor cell migration, proliferation, invasion, and attract lymphocytes especially B cells in vitro. In addition, EBV-immortalized B cells can also be attracted by CCL28. On the other hand, EBV could infect the CCL28 transfectants and replicate in the cells. However, in SCID mice bearing CCL28 transfected NPC xenograft, the tumor growth and metastatic activity were slightly to moderately increased by this gene, but it has failed to attract mouse lymphocytic infiltration, probably due to the dissimilarity of CCL28 molecular structure between human and mouse species. Also, cDNA microarray analysis of CCL28 overexpressed NPC cells showed a group of oncogenes up-regulated and some oncosuppressor genes down-regulated. It is concluded that CCL28 gene may play a role to promote the formation of lymphoepitheliomatous carcinoma of nasopharynx (LE-NPC) and as an oncogene in NPC pathogenesis to promote LE-NPC migration, proliferation and invasion in addition to it chemotactic property. | en |
dc.description.provenance | Made available in DSpace on 2021-06-17T00:16:23Z (GMT). No. of bitstreams: 1 ntu-101-R99444001-1.pdf: 9022584 bytes, checksum: b1c182662c2f96bbc1f5f6dda5d25f0f (MD5) Previous issue date: 2012 | en |
dc.description.tableofcontents | 口試委員審定書 i
致謝 ii 中文摘要 iii Abstract v List of Figures ix List of Tables xi Introduction 1 1. Nasopharyngeal carcinoma (NPC) 1 2. Etiology of NPC 4 3. Molecular biomarkers and prognostic factors of NPC 9 4. SRY (sex-determining region Y)-box 5 (SOX-5) 12 5. Chemokine (C-C motif) ligand 28 (CCL28) 14 6. CCL28 and cancer 16 Materials and Methods 18 1. Cell lines 18 2. Extraction of RNA and preparation of cDNA 19 3. Quantitative RT-PCR (qRT-PCR) 20 4. Statistical analysis of qRT-PCR results 21 5. Enzyme-linked immunosorbent assay (ELISA) 21 6. Immunohistochemical staining. 22 7. Gel extraction 24 8. DNA ligation 25 9. Amplification of plasmids 26 10. Maxi-plasmids purification 27 11. Establishment of a stable NPC cell line transfected by tet-on plasmid containing CCL28-cDNA (pBIG2i-CCL28) 28 12. Scratch wound healing assay 29 13. MTT Assay 29 14. Invasion assay 30 15. Isolation of peripheral blood mononuclear cell 31 16. Chemotaxis assay in transwells and cell staining 31 17. Preparation of EBV particles 32 18. Procedure for EBV infection 33 19. In vivo assay of xenograft growth 34 20. Microarray analysis 35 21. Statistical analysis 35 Results 36 CCL28 gene expression in NNM and NPC tumor cell lines 36 CCL28 protein expression in NPC biopsy specimens 36 Construct plasmid containing CCL28-cDNA (pBIG2i-CCL28) 37 Establishment of the stable pBIG2i-CCL28 transfected NPC cell line 38 The relationship of SOX5 and CCL28 expression 39 The relationship between CCL28 expression and NPC prognosis 39 Functional analysis of CCL28 gene expression 40 (1) CCL28 can enhance the migration rate of NPC cells 40 (2) CCL28 can enhance the proliferation rate of NPC cells 41 (3) CCL28 can enhance the invasion activity of NPC cells in vitro 41 CCL28 can attract the B cell migration to tumor cells 42 Immunohistochemical staining of T cell and B cell in NPC biopsy specimens 42 EBV-immortalized B cells can be attracted by CCL28 43 EBV can infect CCL28-overexpressed NPC-TW01 cells 43 Functional analysis of CCL28 in SCID mice bearing NPC xenografts 44 Mouse mononuclear cells cannot be recruited by CCL28 45 cDNA microarray analysis of mRNA expression in CCL28 transfected NPC cells 45 Discussion 47 Figures 54 Tables 80 References 82 | |
dc.language.iso | en | |
dc.title | CCL28基因在淋巴上皮性鼻咽癌之功能分析 | zh_TW |
dc.title | Functional Analysis of CCL28 Gene in Lymphoepitheliomatous Carcinoma of Nasopharynx | en |
dc.type | Thesis | |
dc.date.schoolyear | 100-2 | |
dc.description.degree | 碩士 | |
dc.contributor.coadvisor | 林欽塘(Chin-Tarng Lin) | |
dc.contributor.oralexamcommittee | 林中梧(Chung-Wu Lin),陳美如(Mei-Ru Chen),施修明(Hsiu-Ming Shih) | |
dc.subject.keyword | 鼻咽癌,淋巴上皮性鼻咽癌,CCL28,SOX5,EBV,建構pBIG2i-CCL28質體,CCL28之體內外功能分析, | zh_TW |
dc.subject.keyword | NPC,lymphoepitheliomatous carcinoma of nasopharynx (LE-NPC),Chemokine (C-C motif) ligand 28 (CCL28),SRY (sex-determining region Y)-box 5 (SOX-5),EBV,Construction of an inducible CCL28 plasmid (pBIG2i-CCL28),Function analysis of CCL28 in vitro and in vivo, | en |
dc.relation.page | 91 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2012-07-03 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 病理學研究所 | zh_TW |
Appears in Collections: | 病理學科所 |
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