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研究生:陳月英
研究生(外文):Yueh-Ying Chen
論文名稱:子宮頸癌及其前驅病變病灶中人類乳突病毒基因型 分析及環氧化酶-2表現
論文名稱(外文):Analysis of HPV genotype and cyclooxygenase-2 expression in uterine cervical cancer and precancerous lesions
指導教授:周寬基周寬基引用關係
口試委員:邱繡河蘇柏誠
口試日期:2010-07-10
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生命科學院碩士在職專班
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:中文
論文頁數:47
中文關鍵詞:子宮頸癌人類乳突病毒環氧化酶-2
外文關鍵詞:uterine cervical cancerHPVCOX-2
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目的: 子宮頸癌的死亡率高,必須早期發現及治療;感染人類乳突病毒是子宮頸癌之重要原因及必要條件,因此人類乳突病毒篩檢及疫苗推廣俱為重要;本研究即在探討子宮頸組織切片中,人類乳突病毒之基因型分析,據以評估上市疫苗之涵蓋比例,並與發炎指標環氧化酶-2等標誌之免疫組織化學染色表現程度做相關性之研究。
材料與方法:自2004-2009年之子宮頸病理檢體,選取141例,病變程度為發炎10例、輕度異生25例、中度異生9例、重度異生14例、原位癌52例、及鱗狀上皮癌31例,做聚合體鏈狀反應引導核酸序列分析,再用基因晶片作HPV基因分型。為了確認HPV是否存在於病變細胞核,選取10例以原位雜合法來查證;141例石蠟包埋檢體,作免疫組織化學染色偵測其環氧化酶-2蛋白的表現。
結果:141例檢體中,偵測到HPV DNA有98例(69.50%,98/141),其中發炎、輕度異生、中度異生、重度異生、原位癌、及鱗狀上皮癌之HPV陽性率分別為40.00%、44.00%、44.44%、78.57%、84.62%及77.42%。HPV陽性例有78例(79.59% 78/98)為單一型感染,20例(20.41%,20/98)為多重感染;單一基因型感染陽性率依序為16 (23.47%)、58 (13.27%)、52 (10.20%)、18 (5.10%)、70 (4.08%),涵蓋56.12%;多重感染(有2-4種基因型)例中,有95%分布於原位癌及鱗狀上皮癌。在98例HPV陽性檢體中,有74例(75.51%)環氧化酶-2染色為陽性,24例(24.49%)環氧化酶-2染色為陰性,而在43例HPV陰性檢體中,有30例(69.77%)環氧化酶-2染色為陽性,13例(30.23%)環氧化酶-2染色為陰性,HPV感染與環氧化酶-2染色表現並無顯著差異(p=0.476),具正相關性(R=0.479)。
結論:目前上市四價疫苗為HPV 6、11、16、18型基因重組疫苗於本研究結果顯示有33.67%的涵蓋率,若能加上宜蘭婦女盛行率前五名之HPV 58、52及70等型別,則預防子宮頸癌涵蓋層面更廣。HPV感染與環氧化酶-2染色並無顯著差異,推測HPV感染與子宮頸發炎及癌化有關,而與疾病的惡化及腫瘤生長因子(環氧化酶-2)沒有顯著差異。
Objective: Uterine cervical cancer has high mortality and should be early detected, diagnosed and treated for the purpose of better prognosis. At present, human papillomavirus (HPV) is the most important and necessary cause of cervical cancer. Therefore, HPV detection in cervical specimens and HPV preventive vaccination are important in cancer prevent. This study is aimed at the HPV genotype survey in Taiwan to predict the efficiency of HPV vaccination. COX-2, as an inflammatory indicator, is also stained by immunohistochemical method to evaluate the correlation with HPV infection.
Materials and methods: One hundred and forty-one paraffin blocks were enrolled for the HPV genotyping by EASYCHIP HPV Blot and COX-2 immunohistochemistry stain. Results: In the 141 cases, 98 cases (98/141,69.50 %) were positive for HPV DNA , as inflammation: 40.00 % , mild dysplasia: 44.00 % , moderate dysplasia: 44.44 % , severe dysplasia: 78.57 % , CIS: 84.62 %, cancer: 77.42%. In 98 HPV DNA positive cases, 78 cases (79.59%) had single type infection and 20 cases (20.41%) had multiple type infection. The first five single genotypes were HPV 16 (23.47%), 58 (13.27%), 52 (10.20%), 18 (5.10%) and 70 (4.08%), respectively and were 56.12 % in total. In multiple type infection, 95 % had lesions of CIS or cancer. In the 98 HPV positive cases, COX-2 IHC stains show 74 positive stain (75.51 %), 24 negative stain (24.49%). In the 43 HPV-negative cases, 30 cases (69.77%) show COX-2 positive stain and 13 cases (30.23%) show negative stain.
Conclusion: The commercial HPV vaccine covers genotypes 6, 11, 16, 18 but not covering the first five genotype, such as 52, 58, 70, in Taiwan (covering rate was 33.67 %). There is no significant correlation between the HPV and COX-2, suggestive of HPV infection was related to cervical inflammation and the succeeding carcinogenesis but not strongly related to the tumor progression and tumor growth factors (COX-2).
目次
中文摘要 ………………………………………………………………I
英文摘要………………………………………………………………II
目次 …………………………………………………………………III
圖表目次………………………………………………………………IV
緒論 ……………………………………………………………………1
第一章 文獻探討 ……………………………………………………2
一、子宮頸癌之相關重要查驗因子:人類乳突病毒、環氧化酶-2 …2
二、研究動機與目標 …………………………………………………6
第二章 材料與方法……………………………………………………7
一、子宮頸組織檢體來源 ……………………………………………7
二、免疫組織化學染色抗體來源 ……………………………………7
三、切片染色 …………………………………………………………7
四、EASYCHIP HPV Blot………………………………………………8
五、免疫組織染色法…………………………………………………11
六、原位雜合反應……………………………………………………12
七、IHC染色之玻片評估 ……………………………………………12
八、統計方法…………………………………………………………12
第三章 結果………………………………………………………… 13
一、子宮頸發炎、異生、原位癌及侵襲癌檢體中EASYCHIPHPV Blot檢查結果……………………………………………………… 13
二、各種子宮頸病變檢體中,HPV感染與COX- 2、年齡、Ki-67、p16及p53表現之相關性 ……………………………………………14
第四章 討 論 ………………………………………………………15
第五章 結 論 ………………………………………………………18
參考文獻………………………………………………………………34

圖表目次
表一 Primer sequences ……………………………………………19
表二 HPV感染與不同子宮頸採樣方式之病變結果分析……………20
表三 HPV單基因型在不同子宮頸採樣方式之病變結果分析………21
表四 HPV多基因型在不同子宮頸採樣方式之病變結果分析………22
表五 單基因型HR-HPV、LR-HPV感染與子宮頸病變程度之相關性 23
表六 HPV感染基因型與年齡分布……………………………………24
表七 HPV多基因型感染與年齡分布…………………………………25
表八 子宮頸病變之HPV感染與各參數之比較………………………26

圖一 子宮頸癌病例COX-2 IHC染色各種染色強度(+3,+2,+1,0)之判讀 …………………………………………………………27
圖二 子宮頸病灶切片HPV單一基因型陽性率前五排名……………28
圖三 子宮頸不同病變程度HPV陽性率分布…………………………29
圖四 子宮頸原位癌及子宮頸癌之HPV單一基因型陽性率前五排名30
圖五 HPV基因型陽性率前五排名之年齡分布………………………31
圖六 ISH染色顯示HPV存在於子宮頸病變細胞核內(200X) ………32
圖七 子宮頸不同病變程度之COX-2陽性率分布……………………33

附錄圖一 HPV的基因組結構…………………………………………44
附錄圖二 HPV感染子宫頸基底細胞並嵌入細胞核導致子宫頸癌的癌生成過程 …………………………………………………45
附錄圖三 HPV E6及E7與p53及pRb作用機制 ………………………46
附錄圖四 Papillomavirus Phylogeny (L1 DNA Sequence)依DNA序列相似度之HPVs分型樹狀圖(親緣圖) ……………………47
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