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研究生:李致叡
研究生(外文):Chih-Jui Lee
論文名稱:偵測子宮頸癌病人中人類乳突瘤病毒血清型第18型E7抗原與抗E7抗體的表現
論文名稱(外文):Detection of the HPV-18 E7 antigen and anti-E7 antibody in human cervical cancer
指導教授:黃光裕
指導教授(外文):Guang-Yuh Hwang
學位類別:碩士
校院名稱:東海大學
系所名稱:生命科學系
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:2006
畢業學年度:94
語文別:中文
論文頁數:60
中文關鍵詞:人類乳突瘤病毒子宮頸癌抗E7抗體
外文關鍵詞:HPVCervical canceranti-E7 antibody
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全球的癌症婦女內有12%為子宮頸癌。 在西元2004年台灣的統計當中,子宮頸癌排名十大癌症死亡原因的第七名,同時亦是女性癌症十大癌症死亡原因的第五名。 根據許多研究顯示出子宮頸癌患者有超過90%以上含有人類乳突瘤病毒核酸,其中最常見的是血清型第16型和血清型第18型。 E6和E7致癌蛋白質,兩種非結構性病毒蛋白,對於細胞的轉型已有很完整的了解,而L1蛋白質為病毒外殼結構蛋白質。 因此選定此三個的抗原,作為血清學研究的依據。 本實驗室已對25位病患經臨床診斷罹患子宮頸癌的病患,確認感染人類乳突瘤病毒血清型第18型,並且於病灶組織偵測L1和E6抗原已有初步的結果。 目前血清學的研究對第18型的了解較不齊全,因此針對第18型進行血清學的研究。 利用免疫酵素連結吸附檢測法測量25位病患和22位正常個體血清抗E6和抗L1抗體之研究亦有初步的結果。 因此本實驗將針對人類乳突瘤病毒血清型第18型中的非結構性蛋白質E7進行探討。 E7蛋白質在病毒生活史中病毒生產時期扮演重要的角色。 此外E7蛋白質可以和pRb相互作用,解除對細胞週期的調控,造成細胞的轉型。 利用西方墨點法已偵測到多數子宮頸癌病人腫瘤組織和相對正常組織含有血清型第18型E7抗原的表現。 另外,使用穀胱苷肽轉移酶接合系統表達選定的病毒蛋白,而表達出的GST-E7蛋白質作為抗原來偵測先前提及的血清其抗GST-E7抗體的結合能力。 25位病患抗GST-E7的表現顯著高於22位正常個體。 這些結果說明受到人類乳突瘤病毒感染的子宮頸癌病患中,不論在病人的病灶位置或是相對正常位置皆顯示出E7的抗原表現。 另外也觀察出子宮頸病患血清中具有較高結合能力的抗血清型第18型E7非結構性蛋白質抗體,可能做為未來研究潛在的診斷標記分子。
Cervical cancer accounts for almost 12% of all cancers in women globally. The statistical information in 2004, cervical cancer is the seventh leading cause of death in Taiwan, and the fifth leading cause of death among major cancers in women. According to the previous studies, at least 90% of the cervical cancer patients contain human papillomavirus (HPV) DNA, and types -16 and -18 are the most common one of HPV. Therefore, we will focus on the HPV-18 serological study. E6 and E7 oncoproteins, two viral nonstructural proteins, are involved in cell transformation, and L1 is the viral structural protein. These three antigens were selected for serological studies. Our laboratory had detected HPV-18 L1 and E6 antigens in cervical tissue from 25 patients with clinically confirmed cervical cancer and the HPV serotype was identified as the type 18. Currently, the serological information of HPV-18 was rare. The binding of the anti-E6 and anti-L1 antibody from 25 patients and 22 normal individuals had been measured by enzyme-linked immunosorbent assay previously. In this study, we focus on the nonstructural protein, the E7 protein of HPV-18. The E7 plays a critical role in the productive stage of the viral cycle. It also can interact with pRb resulting in abrogation of cell cycle that caused cell transformation. In my works, most patients expressed the E7 antigen in tumor tissue and their normal counter part assayed by Western blot. We also used glutathione S-transferase (GST) fusion system to express the HPV-18 E7 viral proteins in E. coli. The GST-E7 fusion protein was used as antigens to measure the binding of anti-E7 antibody in sera. The expression of anti-E7 antibody in 25 patients is significant higher than the 22 normal individuals. These result shows both in patients’ tissue part and normal counter part containing the E7 antigen. Also, the higher binding of anti-E7 antibody observed in the patients’ sera indicated that the anti-E7 antibody may also be a potential marker for diagnosis in the future.
目錄
目錄------------------------------------------------------- 1
中文摘要--------------------------------------------------- 3
英文摘要--------------------------------------------------- 4
前言------------------------------------------------------- 5
人類乳突瘤病毒感染與環境因素---------------------------- 5
人類乳突瘤病毒的特性------------------------------------ 5
人類乳突瘤病毒的分類------------------------------------ 6
人類乳突瘤病毒生活史------------------------------------ 7
人類乳突瘤病毒致病機制---------------------------------- 7
人類乳突瘤病毒引發的免疫機轉---------------------------- 8
人類乳突瘤病毒血清學研究-------------------------------- 10
材料與方法-------------------------------------------------- 13
子宮頸癌組織與血清-------------------------------------- 13
穀胱苷肽轉移酶基因接合係統------------------------------ 13
聚合酶連鎖反應-------------------------------------- 14
勝任細胞的備製-------------------------------------- 15
質體重組與轉型作用---------------------------------- 15
重組質體的純化與濃度的檢測-------------------------- 16
重組質體方向性的確認-------------------------------- 17
重組蛋白質的表達------------------------------------ 17
重組蛋白質的純化------------------------------------ 18
子宮頸癌組織蛋白質的萃取-------------------------------- 19
蛋白質濃度的測定---------------------------------------- 19
蛋白質電泳分析------------------------------------------ 20
蛋白質電泳膠體染色-------------------------------------- 20
西方轉漬法---------------------------------------------- 21
酵素連結免疫吸附檢測法---------------------------------- 23
組織染色------------------------------------------------ 23
免疫組織染色---------------------------------------- 23
結果-------------------------------------------------------- 25
一、子宮頸組織HPV-18 E7抗原的研究------------------------ 25
免疫組織染色---------------------------------------- 25
西方點墨法------------------------------------------ 25
二、聚合酶酵素連鎖反應與重組質體方向性確認--------------- 25
三、重組蛋白質的表達與純化------------------------------- 26
四、子宮頸癌病人中抗HPV-18 E7抗體的研究------------------ 27
討論-------------------------------------------------------- 28
參考文獻---------------------------------------------------- 34
圖表目錄---------------------------------------------------- 40
結果(圖)---------------------------------------------------- 41
附圖---------------------------------------------------- 54
附表---------------------------------------------------- 58
個人資料---------------------------------------------------- 60
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