最近本研究室之研究結果顯示，人類乳突病毒 (human papilloma- virus, HPV) 16和18型之 E6致癌蛋白確實會表現於肺腫瘤組織中，且會參與 p53蛋白之去活化。 本研究擬探討人類端粒酶反轉錄酶 (human telomerase reverse transcriptase, hTERT ) 之轉錄是否會經由 E6致癌蛋白所活化？ 並了解 E6致癌蛋白是否會誘發 hTERT之表現，而參與肺腫瘤之發展。 本研究以135個肺癌患者之腫瘤檢體以免疫組織化學分析法 (immunohistochemistry, IHC) 偵測HPV16/18 E6致癌蛋白表現，並以同步反轉錄聚合酶連鎖反應 ( Real time RT-PCR) 與原位雜交法 (In Situ Hybridization, ISH) 分析 hTERT mRNA之表現，是否會受到 E6致癌蛋白表現之影響？ 結果發現有 HPV16/18 E6致癌蛋白表現之肺腫瘤的 hTERT mRNA表現量，顯著高於沒有 E6致癌蛋白表現之肺癌腫瘤，尤其是女性、不抽菸以及肺腺癌患者之 hTERT mRNA表現量高於男性、抽菸和麟狀上皮癌之患者。另外，在早期 (第I期) 肺癌患者的 hTERT mRNA量亦較晚期 (第II、III期) 患者高。
為了解 E6致癌蛋白是否參與 hTERT之轉錄活化？本研究以小片段核糖核酸干擾技術 (RNAi)， Western Blot和核染質免疫沈澱聚合酶連鎖反應 (Chromatin immunoprecipitation CHIP) 等方法，以了解 hTERT 轉錄是否經由 c-Myc和 Sp1而轉錄調控？ 然後以及時定量聚合酶連鎖反應 Real Time Quantitative Telomeric Repeat Amplification Protocol (Real Time TRAP) 分析有 HPV16 感染之 TL-1肺癌細胞之端粒酶活性，是否會因 E6致癌蛋白 RNAi之轉染而降低？ 同時以軟瓊脂聚落生物分析 (Soft agar assay) TL-1細胞之致癌潛力，是否會因 E6致癌蛋白 RNAi之轉染而降低？核染質免疫沈澱聚合酶連鎖反應結果顯示， Sp1與 c-Myc會協力活化具有 E6致癌蛋白之 TL-1細胞的 hTERT 轉錄作用，這現象同樣能在 HPV16 E6致癌蛋白表現之 SiHa子宮頸癌細胞中觀察到。 若轉染不同劑量的 E6致癌蛋白-RNAi和 hTERT-RNAi，則會明顯降低 TL-1細胞的端粒酶活性。 軟瓊脂聚落生物分析之結果發現， TL-1細胞之致癌的潛力確實在轉染 E6致癌蛋白-RNAi後會顯著降低。 若轉染 hTERT-RNAi之 TL-1細胞株則無法長出穩定之細胞聚落。因此人類乳突病毒 16和 18型之 E6致癌蛋白轉錄活化 hTERT是肺癌化過程的必要條件。
為明白hTERT mRNA表現與肺癌患者之預後是否相關? 我們以 RT-PCR及TRAP方法分析 56個肺腫瘤的 hTERT mRNA表現與端粒酶活性，並且以Kaplan-Meier method分析患者之存活曲線顯示： 沒有端粒酶活性之肺癌患者存活期，較有端粒酶活性的患者為長，且具有統計上的顯著差異 (P < 0.05)。 但是hTERT mRNA表現與否則與患者之臨床預後沒有相關性。 因此再活化之端粒酶活性或許能做為非小細胞肺癌 (non-small cell lung cancer, NSCLC) 之臨床預後因子。

Our recent report indicates that HPV16/18 E6 oncoprotein is expressed in lung tumors and is related to p53 inactivation. We further explored whether hTERT transcription could be upregulated by E6 and contributes to lung tumor development. In the present study, HPV16 E6 oncoprotein of 135 lung tumors was detected by immunohistochemistry (IHC), and hTERT mRNA was evaluated by real-time RT-PCR and in situ hybridization, respectively. Our data indicated that hTERT mRNA levels in E6 -positive tumors, which were prevalent in female nonsmokers and adenocarcinomas, were significantly higher than in E6 -negative tumors. In addition, hTERT mRNA levels in early tumors (stage I) were greater than levels in advanced tumors (stage II and III). CHIP assay showed that Sp1 cooperated with c-Myc to activate hTERT transcription in TL-1 cells which was similar to the SiHa cells. The telomerase activity of the TL-1 cells decreased concomitantly with the transfection of various doses of E6 or hTERT-RNAi. The telomerase activity and oncogenic potential of TL-1 with or without E6 or hTERT-RNAi was determined by real-time quantitative TRAP ASSAYanalysis and soft agar assay, respectively. A soft agar assay showed that the oncogenic potential of TL-1 cells was significantly reduced after being transfected with E6 RNAi. Moreover, the oncogenic potential of TL-1 cells was almost diminished after being transfected with hTERT RNAi. Collectively, transcriptional activation of hTERT by E6 is required for HPV16/18-infected lung tumorigenesis. To verify whether hTERT mRNA and telomerase activity could predict the clinical outcome of lung cancer, 56 lung tumors were enrolled to determine hTERT mRNA and telomerase activity by RT-PCR and TRAP ASSAYassay, respectively. Kaplain-Meier analysis showed that patients without telomerase activity had favorable survival than those with telomerase activity； however, the prognostic significance was not observed in hTERT mRNA. Therefore； reactivated telomerase activity may be a poor prognostic factor in non-small cell lung cancer.

謝誌
中文摘要 1
英文摘要 4
第壹章 研究背景 6
一、人類乳突瘤病毒 (Human Papillomavirus; HPV) 的種類 6
二、 HPV的致癌機轉 11
1. HPV的感染及複製 11
2. HPV E6蛋白與 p53 抑癌基因 13
3. HPV E7 蛋白與 Rb 抑癌基因 15
三、人類乳突瘤病毒與肺癌形成之相關性 16
1. HPV與其他癌症的相關研究 16
2. HPV與肺癌的相關研究 18
四、人類端粒酶反轉錄酶
(human telomerase reverse transcriptase; hTERT) 20
1. hTERT的構造與功能 20
2. hTERT和癌症的相關性 22
3. hTERT與HPV感染之相關性 24


第貳章 研究目的 26
第參章 材料與方法 29
1. 肺癌患者之腫瘤組織之收集 29
2. RNA萃取 29
3. RNA之純化 30
4. RT-PCR (Reverse Transcriptase Polymerase Chain Reaction) 30
5. 及時定量聚合酶連鎖反應(Real Time Quantitative RT-PCR) 31
6. 染色體端粒酶重覆增幅分析
（Real Time Quantitative Telomeric Repeat Amplification Protocol）
( Real Time TRAP) 33
7. 石蠟包埋檢體之製備 34
8. H ＆ E染色 (Hematoxylene ＆ Eosin stain) 34
9. 免疫組織化學染色 (Immunohistochemistry; IHC) 35
10. hTERT 之原位雜交試驗 (In situ hybridization) 35
11. 細胞培養 36
12. RNAi抑制HPV 16 E6或 hTERT基因表現 37
13. 蛋白萃取及西方點墨法 37
14. 核染質免疫沈澱聚合酶連鎖反應
(Chromatin Immunoprecipitation) ( CHIP Assay) 38
15. 軟瓊指菌落形成分析 (Soft Agar Assay) 41
16. 統計分析 42
第肆章 研究結果 43
一、有HPV16/18 E6蛋白表現之腫瘤的hTERT mRNA
表現量顯著高於沒有E6蛋白表現之腫瘤 43
二、Sp1與c-Myc共同合作活化HPV E6蛋白表現
肺癌細胞之 hTERT 基因轉錄作用 45
三、端粒酶的活性與E6蛋白活化 hTERT mRNA
表現有關，會促進TL-1細胞之致癌潛力 47
四、腫瘤組織和肺癌細胞中之hTERT mRNA表現量
與端粒酶活性具有正相關性 48
伍、肺腫瘤組織之端粒酶活性可用來評估
肺癌患者之臨床預後 50
第伍章 討論 51
第陸章 參考文獻 59
第柒章 表與圖 76

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