臺灣博碩士論文加值系統

子宮頸癌是第二常見的女性癌症，而人類乳突病毒（human papillomavirus; HPV）被認為是導致子宮頸癌的一個重要因子，其中又以HPV-16型以及HPV-18型為主要致癌的兩種血清型。由本實驗室之前的研究，我們認為HPV-18在台灣具有盛行率。而HPV的早期基因產物被認為可以跟細胞中某些重要蛋白質作用而阻斷其正常功能，其中E6以及E7蛋白質更是與子宮頸癌的發生有密切關連。E6蛋白質可以與抑癌因子p53作用並進而使其降解；E7蛋白質可以與pRb蛋白質結合，使E2F-pRb複合體分離，而導致細胞基因的轉錄活化。在此報告中，第一部份是從子宮頸癌病患組織中尋找出HPV-18的變異體。25位子宮頸癌患者經由臨床確診並取得檢體，其HPV的感染類型是藉由PCR以及南方墨漬法加以確認，結果發現HPV18之感染盛行率達到100％，而在這25位患者中有7位共同感染了HPV-16（28％）。嵌入患者基因體的HPV-18 E6基因片段則利用PCR增幅並選殖出來，定序後取得其序列。在分析後發現25位患者都具有同一靜默變異，位於E6開放讀碼區第183個鹼基，由C變成G。因此在這一部份，我們得知受檢測的25位患者都具有HPV-18的感染，與世界性的感染盛行率不同，我們同時發現這些患者中增幅出來的HPV-18 E6有一個特有的靜默變異位於位於E6開放讀碼區第183個鹼基，由C變成G。另外，藉由組織學的研究，我們也認為HPV-18與腺癌具有高度相關性，顯示HPV-18在某些細胞型態中具有專一性。因此，本報告的第二部份是嘗試將影響HPV-18在不同細胞中表現差異的因子找出。過去的研究指出HPV-18以及HPV-16在不同細胞中具有不同的表現特異性，受到細胞轉錄因子經由病毒基因的上游調控區（upstream regulatory region; URR）進行精密的調控。為了驗證HPV-18及HPV-16型上游調控區在不同細胞間的表現特異性，我們將全長之病毒之上游調控區構築到含有報導基因之無啟動子載體，再將之轉染至HeLa、NIH 3T3以及U2OS三個細胞株並測量其報導基因活性，結果顯示病毒之調控區在不同細胞間呈現了不同的活性。我們同時構築、並轉染了系列刪減之病毒上游調控區片段，發現在HPV-18及HPV-16型上游調控區的5’端存在一個靜默子的區域，而且發現HPV-18 URR之潛在靜默子在U2OS細胞株中表現極大的抑制能力。而為了知道有哪些細胞因子參與其中，我們設計，並合成了數對核酸序列進行核酸-蛋白質結合反應，並藉由電泳飄移試驗（EMSA）來分析，由結果得知有兩個主要的結合區域，一個是中央的增強子區域，另外一個則是被我們認為是潛在靜默子的5’端區域。為了得到直接的分子證據，我們也利用biotin標定之HPV-18型 5’端URR片段與三種細胞株之核蛋白質進行結合反應以純化結合在這些片段上之細胞轉錄因子，同時利用這些純化核蛋白質與寡核酸片段反應，由飄移測試的結果後我們認為TBP、CEBP以及YY-1在HPV-18的 5’端URR可能形成轉錄因子複合體，而能抑制其表現活性。而在HeLa以及NIH 3T3中，HPV-18全長URR仍有顯著報導基因活性的原因，可能是因為有另外一個細胞轉錄因子Oct-1與TBP競爭結合位點而造成抑制複合體無法形成，導致在上述兩種細胞中，全長的HPV-18 URR仍能維持活性。

Cervical cancer is the second leading cancer in women worldwide. It has been considered that HPV was a marked factor of cervical cancer. The most common types that lead cervical cancer are HPV-16 and HPV-18. In previous studies, we concluded that the prevalence of HPV-18 is high and consistent in Taiwan. It also has been known that the products of HPV early genes can interact with cellular proteins in disrupting their normal functions. Particularly, overexpressions of the high-risk HPV early genes E6 and E7 were associated with the development of cervical cancer by interrupting the normal functions of p53 and E2F-pRb complex. The first part of this report is the identification of the variants of integrated HPV-18 E6 gene in the prevalence infection of HPV-18 of cervical cancer patients. Twenty-five cervical cancer patients were clinically identified and the biopsies were obtained. The infectious HPV types were identified by PCR and Southern blotting analysis. The prevalence of HPV infection in our 25 cases was HPV-18 (100%) and 7 out of these 25 (28%) cases were co-infected with HPV-16. The DNA fragments of the integrated HPV-18 E6 were amplified by PCR and cloned. The nucleotide sequences were obtained by sequencing. We found the most dominant mutation among 25 tested patients was a silence mutation C183G of the E6 coding region. In conclusion of the first part, the prevalent HPV infectious serotype is HPV-18, which differs from the worldwide prevalent type. We also identified HPV-18 E6 variants had a unique silence mutation located on C183G in E6 coding region. Moreover, we suggest that HPV-18 was highly associated with adenocarcinoma by histological study. This result implicated that HPV-18 showed cell-specificity in some cell types, and therefore the second part of this dissertation is the studies of differential expressions of HPV-18 in cells. Previous studies indicated HPV-18 and HPV-16 exhibited different specificities in various cells that may be controlled by transcriptional factors via the upstream regulatory region (URR). To verify the specificities of HPV-18 and HPV-16 URR in cells, the full-length URRs of HPV-18 and HPV-16 were constructed into the promoter-less vector, and then transfected to HeLa, NIH 3T3 and U2OS cells to investigate activities by luciferase assays. Results showed that the relative activities of viral promoters were far different among cells. We also transfected the serially deleted URR constructs of HPV-18 and HPV-16 into cells. We found a strong silencer on the URR 5' regions in both serotypes, and the potential silencer of HPV-18 URR exhibited the strong inhibition in U2OS. To position the cellular transcription factors involved in cell specificity, we synthesized the oligo nucleotides and performed the electrophoretic mobility shift assay (EMSA) to investigate the binding of transcriptional factors among cells. We observed two major binding regions located on the central enhancer and the 5’-end, silencer regions. To obtain the direct molecular evidences, we also used the biotinylated fragments of HPV-18 URR 5’-end to purify the bound proteins from cell nuclear extracts and performed the shift assay. These results indicated that the binding of TBP, CEBP and YY-1 may form a complex and inhibit the activities in cells. Moreover, our data also implicated the binding of Oct-1 in HeLa and NIH 3T3 may disrupt the formation of inhibitory complex on HPV-18 URR 5’-end by steric competition with overlapped TBP binding site in maintaining parts of activities in HeLa and NIH 3T3 cells.

目錄………………………………………………………………............1
中文摘要…………………………………………………………………4
英文摘要…………………………………………………………………7
前言……………………………… …………………………………… 10
一、 子宮頸癌與人類乳突瘤病毒的關係…………………………10
二、 人類乳突瘤病毒的基本特性…………………………………10
三、過去對於病毒變異體的研究……………………………….….12
三、上游調控區對人類乳突瘤病毒早期基因E6、E7的調控…...13
四、HPV上游調控區的組織專一性表現與細胞轉錄因子的關係.14
材料與方法……………………………………………………………..17
一、引子序列……………………………………………………….17
二、寡核酸序列…………………………………………………….19
三、檢體…………………………………………………………….21
四、初級細胞與細胞株的培養…………………………………….21
五、Genomic DNA的萃取…………………………………………21
六、PCR-Southern blotting………………………………………….22
七、HPV-18 E6序列變異體的確認………………………………..23
八、質體構築……………………………………………………….23
九、信使核醣核酸（messenger RNA）的萃取及RT-PCR……….25
十、細胞轉染（transfection）……………………………………..26
十一、Luciferase assay………………………………………………26
十二、核蛋白質的萃取…………………………………………….27
十三、寡核酸序列的黏合………………………………………….28
十四、電泳飄移測試（Electrophoresis Mobility Shift Assay; EMSA）…………………………………………………………28
十五、以Dynabeads M-280純化DNA結合蛋白質………………28
十六、統計分析……………………………………………………..30
第一部份：台灣子宮頸癌患者中人類乳突瘤病毒之盛行率以及HPV-18 E6變異體之分析……………………………….31
結果……………………………………………………………………..32
一、子宮頸癌患者之人類乳突瘤病毒感染分型…………………..32
二、HPV-18 E6的變異體分析……………………………………..33
討論…………………………………………………………..…………34
一、在台灣子宮頸癌患者之人類乳突瘤病毒18型盛行率較高….34
二、嵌入病患基因體中的HPV-18 E6的變異體………………….37
第二部分：人類乳突瘤病毒18型之5’端上游調控區具有潛在靜默子的功能…………………………………………………….38
結果……………………………………………………………………..39
一、HPV-18早期基因在不同細胞間的表現………………………39
二、HPV-18及-16之上游調控區在不同細胞間的活性…………..40
三、HPV-18及-16 URR系列刪減對於活性的影響………………41
四、與HPV-18 URR作用的細胞因子…………………………….44
五、參與HPV-18 URR 5’端靜默子功能的細胞因子…………….45
討論…………………………………………………………..…………48
一、HPV-18 URR的組織專一性表現………………………………48
二、HPV-18 URR的5’端具有潛在的靜默子………………………50
三、與HPV-18 URR 5’端潛在靜默子作用的細胞因子…………..53
參考文獻………………………………………………………………..57
表目錄…………………………………………………………………..65
圖目錄…………………………………………………………………..66
表說明……………………………………………………………...…...67
圖說明…………………………………………………………………..70
附錄目錄………………………………………………………………..82
附錄說明………………………………………………………………..83
個人資料………………………………………………………………..86

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