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論文中文名稱:以雜交層析法偵測十六型人類乳突腫瘤病毒感染細胞(SiHa)之研究 [以論文名稱查詢館藏系統]
論文英文名稱:Detection of HPV16 infected cell(SiHa) using hybrid chromatography [以論文名稱查詢館藏系統]
院校名稱:臺北科技大學
學院名稱:工程學院
系所名稱:生物科技研究所
畢業學年度:99
出版年度:100
中文姓名:邱建升
英文姓名:Chien-Sheng Chiu
研究生學號:97688024
學位類別:碩士
語文別:中文
口試日期:2011-01-24
論文頁數:68
指導教授中文名:侯劭毅
口試委員中文名:王勝仕;黃志宏
中文關鍵詞:人類乳突腫瘤病毒雜交層析法奈米金粒子
英文關鍵詞:human papillomavirus 16hybrid chromatographycolloidal gold
論文中文摘要:子宮頸癌(cervical cancer)為女性常見癌症,目前已證實人類乳突腫瘤病毒(human papillomavirus)的感染是導致子宮頸癌生成原因,其中以第16、18型(HPV16、HPV 18 )為最主要。本研究主要目的利用雜交層析法原理來偵測人類乳突腫瘤病毒第16型病毒所感染的細胞。
本實驗中所培養之細胞株SiHa cell 為感染human papillomavirus 16型(HPV 16)之子宮上皮細胞。利用奈米金粒子(colloidal gold )與硫醇(thiol)之間有強大的共價鍵結力,在DNA probe上修飾硫醇與奈米金鍵結形成一個金奈米探針後去雜交( Hybrid )捕捉SiHa cell中的chromosome DNA L1 region再與標有 biotin之探針雜交後與卵蛋白(avidin)結合,利用免疫層析法膜上固定抗生物素卵蛋白之抗體(anti avidin antibody)去捕avidin。則可使奈米金粒子結合在測試區並且形成肉眼可辨識之紅色的線段來偵測目標物。實驗結果顯示偵測雙股DNA可至 2 amole。
論文英文摘要:Cervical cancer is a common cancer in female. Human papillomavirus (HPV) infect has been demonstrated to cause cervical cancer. The type 16 and 18 is the riskiest type among all type HPV types . The purpose of this study is to detect chromosomal DNA which was HPV infected using hybrid chromatography.
In a previous study of our lab, biotin-DNA and SH-DNA probes were designed as capture and detection probes and each probe is complementary with different part of L1 region of HPV type 16. The biotin-DNA probe was associated with streptavidin and anti-streptavidin antibody on the nitrocellulose membrane, while the SH-DNA probe was connected to gold nanoparticles using covalent bond. In this study, 2 amole of dsDNA purified from SiHa cells, HPV-16 infected cervical epithelial cells, were detected.
論文目次:摘 要 I
ABSTRACT II
致謝 III
目 錄 IV
表目錄 VI
圖目錄 VII
第一章 緒論 1
1.1 前言 1
1.2 研究動機及目的 2
第二章 研究背景與原理介紹 3
2.1人類乳突腫瘤病毒簡介 3
2.2 現行子宮頸癌檢測技術 3
2.2.1 柏氏細胞抹片檢查 4
2.2.2薄層細胞學檢查(Thin prep pap smear) 4
2.2.3子宮頸螢光檢查(PapSure) 4
2.2.4電腦輔助抹片判讀 (AutoPap &PapNet) 5
2.2.5分子診斷 5
2.3 SiHa cell line簡介 6
2.4奈米金粒子檢測技術介紹 6
2.4.1 膠體金免疫技術應用 7
2.4.2膠體金特性 7
2.4.3膠體金粒子吸收光譜 7
2.4.4奈米金與DNA的結合 8
2.5免疫層析試紙分析法(Immunochromatography) 9
2.5.1檢測原理 11
第三章 實驗方法與步驟 21
3.1 修飾硫醇DNA固定在金奈米粒子表面 23
3.2 細胞培養(Cell culture) 24
3.3 細胞計數 24
3.4 DNA純化 25
3.5 DNA濃度測量 26
3.6 DNA膠體電泳 (Gel Electrophoresis) 26
3.7 物理方法打斷雙股DNA 27
3.8 硝化纖維膜固定avidin-biotin DNA probe、avidin、anti-avidin antibody 27
3.9膜上固定avidin biotin-probe偵測單股HPV DNA之雜交層析法實驗 28
3.10 膜上固定avdin偵測單股HPV DNA之雜交層析法實驗 29
3.11 膜上固定anti avdin antibody偵測單股HPV DNA之雜交層析法實驗 30
3.12 SiHa cell line chromosome DNA 偵測 31
3.13 定量分析 32
3.14實驗設備 33
3.15 實驗藥品 35
第四章 結果與討論 37
4.1膜上固定avidin-biotin DNA probe偵測單股HPV 16 DNA 37
4.2膜上固定avidin偵測單股HPV 16 DNA 39
4.3膜上固定anti avidin antibody偵測單股HPV 16 DNA 41
4.4以物理方式打斷DNA 43
4.5偵測HPV雙股DNA加熱不同時間 44
4.6 SiHa cell line chromosome DNA 之偵測 44
4.7雜交層析法最佳化條件 45
第五章 結論 47
附 錄 52
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