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研究生:謝若屏
研究生(外文):Jo-Ping Hsieh
論文名稱:建立可測量EB病毒致癌蛋白LMP1影響NF-κB路徑的細胞株以發展抗EB病毒藥物的篩選平檯
論文名稱(外文):A cell-based system for screening compounds which can inhibit Epstein-Barr virus oncoprotein LMP-1-mediated NF-kB pathway
指導教授:余兆松
指導教授(外文):Jau-Song Yu
學位類別:碩士
校院名稱:長庚大學
系所名稱:基礎醫學研究所
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:2007
畢業學年度:95
語文別:英文
論文頁數:49
外文關鍵詞:Nasopharyngeal carcinomalatent membrane protein 1nuclear factor kappa B
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Nasopharyngeal carcinoma (NPC) has a higher incidence rate in Asian than in other areas of the world, especially in southern China, Taiwan, and Hong Kong. Epstein-Barr virus infection and the expression of its oncogene product, latent membrane protein 1 (LMP-1) is believed to contribute to the pathogenesis of NPC. NPC could be diagnosed with high sensitivity (91.4%) and specificity (98.3%) by measuring the levels of LMP-1 and EBNA in the nasopharyngeal swabs. LMP-1 is a six trans-membrane protein which can activate multiple vital signaling pathways, such as NF-κB, MAPK, and JNK. Previous studies have shown that EBV-mediated growth transformation is genetically and biochemically linked to LMP-1-mediated activation of NF-κB. Several lines of evidence indicate that NF-κB pathway activated by LMP-1 plays a critical role in NPC progression. Because there is no effective treatment for this disease, we intend to establish a 96-well, high-throughput cell-based assay system to screen for potential compounds, which can block the NF-κB pathway activated by LMP-1. We constructed two plasmids, pGL3-4κB-SEAP and p4κB-DsRed2-N1 to monitor the activation level of the NF-κB transcription factor. The reporting activity was evaluated by known regulators for NF-κB pathway, such as TNF-α and LMP-1 (NF-κB inducers) and EGCG, PDTC and curcumin (NF-κB inhibitors). The SEAP activity is up-regulated by TNF-α and LMP-1 (~3-5 fold activation) and down-regulated by inhibitors (~40% without cytotoxicity). Similarly, in p4κB -DsRed2-N1 transfected cells, the fluorescence intensity was increased by TNF-α and LMP-1 treatment, respectively. These results indicate that both reporter plasmids are responsive to NF-κB activation mediated by LMP-1, with the signal-to-noise ratio suitable for use in high-throughput screening system for drug discovery. Pilot screening with 5500 compounds (in 69 plates) demonstrates that the reporting system is a robust screening assay, with Z values ³ 0.5 for 58 plates. MTS assay was conducted simultaneously to evaluate cytotoxicity of test compounds. Several compounds which exhibit substantial inhibition in the SEAP assay without showing significant cytotoxicity were identified from the screening. Further studies to characterize these compounds are currently underway.
Acknoeledgement……………………………………………………….P1

Abstract……………………………………………………………...P2-P3

Introduction………………………………………………………...P4-P13

Material and Method………………………….…………………..P14-P17

Result…………………………………………………………….P18-P22

Discussion………………………………………………………...P23-P27

Figure……………………………………………………………..P28-P44

Reference…………………………………………………………P45-P49
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