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標題: | Nrf1基因在鼻咽癌之功能分析 Functional Analysis of Nrf1 Gene in Nasopharyngeal Carcinoma |
作者: | Ting-Ying Chen 陳亭螢 |
指導教授: | 林欽塘(Chin-Tarng Lin) |
關鍵字: | 鼻咽癌,NRF1,CRISPR / Cas9, NPC,NRF1,CRISPR/ Cas9,Nude mice, |
出版年 : | 2018 |
學位: | 碩士 |
摘要: | 鼻咽癌為一種上皮細胞癌,好發於中國南方、東南亞、台灣及北非。目前其確實之致病機轉仍不是很清楚,但可能與EB病毒 ( Epstein-Barr virus ) 之感染、環境因子及遺傳因素有關。本論文研究的主要目的是探討鼻咽癌的發生與基因的關係。我們運用cDNA微陣列分析 ( cDNA Microarray Analysis ) 的方法比較鼻咽癌細胞株以及鼻腔內膜上表皮細胞之基因表現的差異,選出了11個在兩者之間有差異表現的基因,然後再經由即時定量聚合酶連鎖反應 ( Quantitative RT-PCR )的分析而發現FGFR1基因及SPARC基因在鼻咽癌細胞中的表現明顯減少;接著我們進一步觀察與FGFR1基因及SPARC基因有關的轉錄因子NRF1 ( Nuclear respiratory factor 1 ),再經由即時定量聚合酶連鎖反應 ( Quantitative RT-PCR ) 和西方點墨法 ( Western blotting ) 的確認分析,及細胞免疫染色法的觀察後,我們發現NRF1基因在鼻咽癌細胞的細胞核有高表現的現象。為了更進一步了解NRF1基因與鼻咽癌的關係,我們利用CRISPR / Cas9的技術將本實驗室所建立的TW01人類鼻咽癌細胞株的NRF1基因剔除,使其NRF1表現量下降,藉此觀察此基因在鼻咽癌病理機制上所扮演的角色及功能。我們首先利用西方點墨法 ( Western blotting ) 確認NRF1基因剔除之鼻咽癌細胞之NRF1蛋白表現量是否有顯著的下降,接著挑選出38株single cell lines,再從中挑選剔除效果最好的細胞株來進行接下來的實驗。我們發現剔除NRF1的鼻咽癌細胞的爬行速度及生長速度均較原本的鼻咽癌細胞株緩慢,且以雙氧水增加細胞氧化壓力的實驗發現剔除NRF1的鼻咽癌細胞較容易死亡,接著於氧氣消耗速率及細胞糖解作用的實驗都發現剔除NRF1的鼻咽癌細胞在此兩種代謝中都有下降的情形。在動物實驗的部分,我們發現打入剔除NRF1鼻咽癌細胞的Nude小鼠其腫瘤生長情形較正常鼻咽癌細胞的Nude小鼠緩慢。由體外的細胞實驗及動物實驗結果,我們推論NRF1在鼻咽癌的形成扮演一種致癌基因的角色。 Nasopharyngeal carcinoma ( NPC ) is arising from nasopharyngeal epithelium. Endemic regions of NPC are shown in southern China, southeast Asia, northern Africa and Taiwan. The specific etiology is not clear now, but some evidences show that NPC is related to environmental factors, heredity, and Epstein-Barr virus infection . The purpose of this research was to find out the genes associated with NPC pathogenesis. Previously we compared the mRNA expression between NPC cell lines and normal nasal mucosal epithelial cells by cDNA microarray analysis, and found that 11 genes showed significantly increased or decreased expression. Then we used quantitative RT-PCR for further confirmation, and found that FGFR1 gene and SPARK gene were significantly decreased in NPC cell lines. In addition, we also found that the transcription factor NRF1 ( Nuclear respiratory factor 1 ) could regulate the FGFR1 gene and SPARK gene expression. When we compared NRF1 gene expression between NPC cell lines and normal nasal mucosal epithelial cells by quantitative RT-PCR, Western blotting and immunocytochemistry, we found that NRF1 gene was significantly increased expression in NPC cell lines. In order to understand the role of NRF1 expression in nasopharyngeal carcinoma, we used CRISPR/Cas9 to knockout NRF1 gene in NPC TW01 cell line which was established from our lab. After knockout of NRF1, we used Western blotting to confirm the NRF1 expression in NPC cells, and found that the NRF1 protein was significantly decreased. Then we produced 38 single stable cell lines, and chose the best gene knockout clone for the following experiments. We found that NRF1 knockout could decrease cell proliferation, migration, and invasion activities of NPC cells. When we added hydrogen peroxide into medium to increase oxidative stress, we found that NPC cells which expressed lower NRF1 had worse viability. As for metabolism, we found that both of mitochondrial respiration ability and glycolysis ability were downregulated in knockout of NRF1 cells. The in vivo animal experiment by injection of NRF1 knockout cells also revealed that the tumors growth was decreased, which confirmed the results of in vitro assay. According to in vitro assay and xenograft experiment, we suggest that NRF1 plays a role as an oncogene in nasopharyngeal carcinoma pathogenesis. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/71247 |
DOI: | 10.6342/NTU201801833 |
全文授權: | 有償授權 |
顯示於系所單位: | 病理學科所 |
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