毒殺型T淋巴球(CTL)免疫反應被認為是可以有效清除腫瘤細胞的生長、限制病毒的擴散與清除病毒感染。利用單一個或多個抗原決定位的疫苗已經有成功的發展，而且結果也可有效的引發具抗原決定位特異性的CTL免疫反應可用來對抗各種致病源與癌症。針對同一個蛋白質可能有一連串不同的CTL抗原決定位，這是因爲不同的HLA基因型所造成的差異性；而HLA-A24是亞洲人口中最多的HLA基因型之一，尤其在台灣佔有相當高的比率。本論文欲生產HLA-A24基因轉殖鼠，來作為疫苗開發等相關試驗的實驗平台；利用microinjection的技術將構築完成且確定可以表現的H-2Kb P/A24及H-2Kb P/A24-Kb質體注射至C57BL/6小鼠受精卵，再以PCR及流式細胞儀檢驗轉殖基因在小鼠體內表現與否。
人類乳突瘤病毒(Human Papilloma Virus，HPV)之感染與子宮頸癌的發生有直接的相關性，HPV E5蛋白主要存在受感染細胞之胞器的脂質雙層膜上，可能會將MHC class I複合體滯留於細胞內，藉此逃避CTL免疫毒殺反應。本論文欲利用合成胜肽片段E5 11-52及E5 59-83來生產抗HPV-16 E5抗體，用以觀察子宮頸癌患者病理切片中E5蛋白之表現，並觀察此病理切片上MHC class I分子的表現，探討E5蛋白是否影響MHC class I分子的表現。
此外還以組織切片染色觀察正常及患者子宮頸切片組織上NRIP及細胞核受體的表現差異。細胞核受體(Nuclear receptors)的主要功能為攜帶荷爾蒙進入細胞核刺激部分特定基因轉錄活性，因此在荷爾蒙相關腫瘤中扮演重要的角色，例如部份肝癌是雄激素(androgen)依賴性腫瘤可以抗雄激素治療；其他的荷爾蒙相關腫瘤還有乳癌與子宮頸癌都被懷疑與體內荷爾蒙失調有關。在2005年發現的新穎蛋白質nuclear receptor interaction protein (NRIP)被發現可以與帶有ligand的雄激素受體(androgen receptor，AR)與糖皮質受體(glucocorticoid receptor，GR)結合促進轉錄活性；此外BCAS2 (亦稱DAM1)在1999年於乳癌細胞株中被發現此段DNA序列特別的被複製放大，之後又發現此一蛋白的過量表現會促進雌激素受體(estrogen receptor，ER)所刺激的轉錄活性，反之則降低。本論文利用共同免疫沉澱法，觀察到無論有無ligand的加入，雄激素受體、糖皮質受體、雌激素受體及黃體素受體皆會與NRIP或BCAS2相互建結。

CTL responses are thought to affect the tumor growth, and the virus spreading during infection. Subunit vaccine have beeen successfully developed by inducing epitope-specific CTLs responses efficiently. These epitopes-based CTLs responses are restricted by HLA alleles. According to previous reports of the frequency of HLA alleles in humans, HLA-A24 is one of the most common HLA-A alleles in Asia. High frequency of HLA-A24 and relatively restricted HLA polymorphisms are noted in Taiwan. In my study, HLA-A24 transgenic mice will be generated and used as preclinical disease models and that provide the information relevant to clinical experiments. The plasmid, H-2Kb P/A24 and H-2Kb P/A24-Kb have been confirm in cell modle. Microinject the transgene into C57BL/6 mice oocyte, and then confirm transgene mice by PCR and FLOW cytometry.
The E5 protein is expressed during the early stages of papillomavirus infected epithelium. The E5 protein is localized in the endo-membrane compartments of lipid bi-layers membrane, such as Golgi apparatus and endoplasmic reticulum. E5 has the interaction with the vacuolar ATPase 16K ductin/subunit c, is deemed responsible for the lack of acidification in organelles. The cause is that E5 arrests the MHC class I complexes in the Golgi apparatus and impedes they were transported to the cell surface.In my study, the specific anti-HPV 16 E5 (a.a.11-52 and a.a.59-83) polyclonal antibody will be generated and used to identify the E5 expression in HPV-16-positive and HPV-16-negative cervical tissues sections. The antibody will also be used to identify the MHC class I within these tissues, that to investigate the correlation between the oncoprotein E5 and MHC I presentation. In order to confirm the E5 protein expression.
Nuclear receptors, such as androgen receptor (AR) and glucocorticoid receptor (GR), play an important role in some hormone-responsive tumors such as cervical cancer. A novel nuclear receptor interaction protein (NRIP) enhances the transcriptional activity of AR or GR in a ligand-dependent manner. And BCAS2 (DAM1) is a novel gene isolated from breast cancer cell line. It was found to be an estrogen receptor (ER) interaction protein by yeast two-hybrid screening. Overexpression of BCAS2 enhanced the estrogen receptor-mediated transcription while inhibition of BCAS2 expression attenuated. Given vital importance of nuclear receptors signaling in hormone-related cervical cancer diseases, we will investigate the role of nuclear receptors and their interaction proteins in cervical cancer diseases development and progression.

標題 頁碼
正文目錄Ⅰ
圖表目錄Ⅵ
中文摘要Ⅶ
AbstractⅨ
第一篇 建立HLA-A24轉殖鼠實驗平台1
第一章 緒論1
第一節 疫苗發展1
第二節 主組織相容性複合體3
第三節 HLA轉殖鼠在胜肽次單位疫苗開發的重要性4
第二章 材料及方法……6
第一節 細胞培養6
壹 293T細胞培養6
第二節 構築轉殖基因6
壹 構築H-2Kb P/A247
一 Insert DNA fragment preparation7
二 pcDNA3-A24 Vector preparation7
三 Ligation7
四 Transformation7
貳 構築H-2Kb P/A24-Kb8
一 Insert DNA fragment preparation8
二 pUC19-H-2Kb intron Vector preparation9
三 Ligation9
四 Transformation9
第三節 確認轉殖基因表現與否…10
壹 細胞轉染表現蛋白10
貳 利用流式細胞儀偵測轉染細胞表現蛋白10
第四節 轉殖鼠體內轉殖基因的確認11
壹 萃取轉殖鼠chromosomal DNA11
貳 聚合酶連鎖反應…12
叁 利用流式細胞儀確認轉殖鼠轉殖基因之表現12
第三章 結果14
第一節 利用流式細胞儀確認H-2KbP/A24及H-2KbP/A24-Kb質體的
表現14
第二節 HLA-A24轉殖小鼠之繁殖與轉殖基因表現確認14
第三節 HLA-A24-Kb轉殖小鼠之繁殖與轉殖基因表現確認15
第四章 討論17
第五章 結論19

第二篇 觀察人類乳突瘤病毒16型E5蛋白以及細胞核受體相關蛋白在
子宮頸癌的表現20
第一章 緒論20
第一節 子宮頸癌與人類乳突瘤病毒之關係20
第二節 HPV與人類子宮頸上皮病變之關係21
第三節 HPV-16 E5 致癌蛋白質22
第四節 荷爾蒙與子宮頸癌的關係24
第五節 Steroid hormone receptor 類固醇荷爾蒙受體之構造與功能24
第六節 NRIP及BCAS2之構造與功能26
第七節 研究策略27
第二章 材料與方法28
第一節 細胞培養28
壹 293T/E5細胞培養28
第二節 抗體之製備28
壹 HA1單株抗體之製備29
一 12CA5融合瘤細胞培養29
二 HA1單株抗體之製備29
貳 HPV-16 E5抗血清之製備29
一 HPV-16 E5 synthetic conjugated peptide (Immunogen)之製備29
二 小鼠生產抗HPV-16 E5抗體實驗30
第三節 利用293T/E5細胞表現之E5蛋白確認抗血清是否呈現抗體
反應31
壹 蛋白質之定量31
貳 免疫沉澱分析法(Immunoprecipitation，IP) 31
叁 SDS聚丙烯醯胺膠電泳法分析 (SDS-PAGE) 32
肆 蛋白質轉漬32
伍 西方點墨法分析 (Western blot) 33
第四節 利用共同免疫沉澱法分析類固醇荷爾蒙受體與NRIP及
BCAS2之間的作用關係33
第三章 結果35
第一節 小鼠免疫注射後之E5抗體確認35
第二節 利用共同免疫沉澱法分析類固醇荷爾蒙受體與NRIP及
BCAS2之間的作用關係35
第三節 觀察子宮頸癌患者組織切片中類固醇荷爾蒙受體與NRIP的
表現情況…37
第四章 討論38
第五章 結論40
參考文獻42
附錄一 BCAS2之DNA序列與胺基酸序列的電腦預測結果
附錄二 材料配方

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