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研究生:劉嘉綺
研究生(外文):Chia-Chi Liu
論文名稱:GPR68基因在鼻咽癌之功能分析
論文名稱(外文):Functional analysis of GPR68 gene innasopharyngeal carcinoma
指導教授:林欽塘林欽塘引用關係
指導教授(外文):Chin-Tarng Lin
口試日期:2017-07-20
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:病理學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2017
畢業學年度:105
語文別:英文
論文頁數:54
中文關鍵詞:鼻咽癌腫瘤發生GPR68CCL28GPR68 之體內外功能分析
外文關鍵詞:NPCTumorigenesisG protein coupled receptor (GPR68)C-C motif chemokine ligand 28 (CCL28)Functional analysis of GPR68 in vitro and in vivo
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鼻咽癌是好發於鼻咽部的惡性腫瘤。它最常見好發於居住在東南亞、新加坡、香港和台灣的華人。鼻咽癌發生的原因不是很清楚但有可能由病毒感染、環境因素和遺傳共同導致的,在過去的研究裡,一般都認為EBV 病毒是鼻咽癌的病源,但最近的文獻顯示EBV 病毒可能只是鼻咽癌進展的因子而不是起始因子,因此,本篇研究的主要目的是要探討基因與鼻咽癌之間的關係。在過去我們實驗室建立了十株鼻咽癌細胞株,我們利用cDNA 微陣列分析正常鼻咽上皮細胞和鼻咽癌細胞內的mRNA 的表現量,並且利用即時定量聚合酶連鎖反應(qRT-PCR)和西方點墨法(Western blotting)確認CCL28 基因很明顯在鼻咽癌細胞表現量較多,我們也針對了CCL28 基因在鼻咽癌所扮演的角色做了研究,並且發現當CCL28 在鼻咽癌
細胞表現高時,GPR68 的表現量會變低。我們也另外建立了另一個shCCL28 之細胞株,利用帶有shRNA 的病毒去感染鼻咽癌細胞使其細胞中的CCL28 的表現量下降,我們發現當CCL28 的表現量下降時,GPR68 的表現量會跟著上升。目前對於GPR68 在鼻咽癌病理機制的影響尚未有相關的研究,因此本篇主要的目的在探討GPR68 在鼻咽癌所扮演的角色。首先,我們利用即時定量聚合酶連鎖反應(qRT-PCR)和西方點墨法(Western blotting) 分析正常鼻咽上皮細胞和鼻咽癌細胞內GPR68 的mRNA 和蛋白的表現量,我們發現GPR68 的mRNA 和蛋白質在鼻咽癌細胞內明顯表現較少,為了更進一步探討GPR68 在鼻咽癌中所扮演的角色,我們將pBIG2i-GPR68 質體轉染(transfect)到NPC-TW01N1 細胞株內,我們發現轉染後NPC-TW01N1 細胞有過度表現GPR68 的蛋白,進而發現,其有顯著的降低鼻咽癌細胞株的生長,爬行和侵襲力。在異種移植的鼻咽癌腫瘤 (NPC xenografts)長於NOD-SCID 之老鼠的實驗中發現以高度表現GPR68 基因的鼻咽癌細胞在NOD-SCID 老鼠體內長到第七週時,腫瘤重量比控制組小,這些結果顯示GPR68可能在鼻咽癌扮演一種抑癌基因的角色, 我們相信未來治療鼻咽癌可藉由抑制致癌基因CCL28,使抑癌基因GPR68 在鼻咽癌中增加表現,達到治療鼻咽癌的效果。
Nasopharyngeal carcinoma (NPC) is one of the most common cancer originating in the human nasopharynx. It is also one of the most common cancers among Chinese living in southern China, Hong Kong, Singapore and Taiwan. NPC is caused by a combination of factors including viral infection, environmental influences, and heredity. It was proposed that Epstein-Barr virus (EBV) is closely associated with NPC pathogenesis; but recent studies have revealed that EBV behaves more likely as a progression factor but not an initiation factor. The aim of this research was to identify the genes associated with NPC pathogenesis. Previously we used cDNA microarray analysis of mRNA expression between NPC cell lines and normal nasal mucosal epithelial cells have identified that, Chemokine (CC motif) of ligand 28 (CCL28) gene expression was significantly increased in NPC cell lines. It was also verified by quantitative RT-PCR and Western blotting analysis. In our studies of the function of CCL28 gene in NPC, we found that G protein coupled receptor (GPR68) was also significantly decreased in NPC cell lines. We also knocked down CCL28 by using shRNA to create a shCCL28 system and found that when CCL28 was down-regulated, the protein expression of GPR68 was increased. The role of GPR68 in the molecular pathogenesis of NPC and its functions is still not clearly reported. Therefore, we analyzed the mRNA and protein levels in the normal nasal mucosal epithelial cells and NPC cell lines by qRT-PCR and Western blotting. We found that both mRNA and protein levels were significantly decreased in NPC cell lines. To further study of the role of GPR68 in NPC, we constructed a stable pBIG2i-GPR68 transfected NPC cell lines. The expression of protein level was significantly increased in the transfected NPC-TW01N1 cell line. This in turn resulted in a significant inhibition of cell proliferation, migration and invasion. In vivo, we have injected overexpression GPR68 NPC cell line to NOD-SCID mice for 7 weeks, the weight of tumor were lower than control group. These results suggest that GPR68 may play a suppressor gene function in NPC pathogenesis.
目錄Table of Contents
口試委員審定書............................................................................................ i
致謝...............................................................................................................ii
中文摘要......................................................................................................iii
Abstract ........................................................................................................ v
目錄Table of Contents..............................................................................vii
List of Figures .............................................................................................ix
List of Tables ................................................................................................ x
Introduction ............................................................................................ - 1 -
Nasopharyngeal carcinoma (NPC) ........................................................................................ - 1 -
Etiology of NPC ................................................................................................................. - 4 -
G protein coupled receptor 68, GPR68 ................................................................................. - 6 -
Chemokine (C-C motif) ligand 28 (CCL28) ......................................................................... - 8 -
Materials & Methods ........................................................................... - 10 -
1. Cell lines .................................................................................................................... - 10 -
2. Extraction of RNA and preparation of cDNA ............................................................ - 11 -
3. Quantitative RT-PCR (qRT-PCR) ............................................................................. - 12 -
4. Statistical analysis of qRT-PCR results........................................................................ - 13 -
5. Immunohistochemical staining .................................................................................... - 13 -
6. Western blotting ........................................................................................................... - 14 -
7. Mini-plasmid purification ............................................................................................ - 15 -
8. Gel extraction ............................................................................................................... - 16 -
9. DNA ligation ................................................................................................................ -17 -
10. Amplification of plasmids .......................................................................................... - 18 -
11. Establishment of a stable NPC cell line transfected by tet-on plasmid containing
GPR68-cDNA (pBIG2i-GPR68) ......................................................................................... - 19 -
12. MTT Assay ................................................................................................................. - 20 -
13. Scratch wound healing assay ...................................................................................... - 21 -
14. Invasion assay ............................................................................................................ - 21 -
15. In vivo assay of xenograft growth .............................................................................. - 22 -
16. Establishment of shCCL28 NPC cell lines by lentiviral vector system ..................... - 22 -
Results .................................................................................................... - 24 -
Knockdown of chemokine (C-C motif) ligand 28 (CCL28) gene can cause over expression of
GPR68 ................................................................................................................................ - 24 -
GPR68 gene expression in NNM and NPC tumor cell lines ............................................... - 24 -
GPR68 protein expression in NPC biopsy specimens ......................................................... - 25 -
Construction plasmid containing GPR68-cDNA (pBIG2i-GPR68) .................................... - 25 -
Establishment of the stable pBIG2i-GPR68 transfected NPC cell line ............................... - 26 -
Functional analysis of GPR68 gene expression in NPC ..................................................... - 26 -
1. GPR68 may inhibit the proliferation rate of NPC cells.............................................. - 26 -
2. GPR68 may inhibit the migration ability of NPC cells .............................................. - 27 -
3. GPR68 can inhibit the invasion activity of NPC cells in vitro ................................... - 27 -
Functional analysis of GPR68 in SCID mice bearing NPC xenografts .............................. - 28 -
Discussion .............................................................................................. - 30 -
Figures ................................................................................................... - 34 -
Tables ..................................................................................................... - 49 -
Reference ............................................................................................... - 51 -
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