臺灣博碩士論文加值系統

至今，子宮頸癌仍可說是婦女常見的惡性腫瘤之一。每年，全世界估計有五十萬的子宮頸癌新病例出現；更有二十萬的婦女因子宮頸癌導致死亡。而人類乳突瘤病毒第十六型就是引發子宮頸癌的元兇之一。其病毒基因中具有E5、E6、E7三種致癌基因，所衍生出來的蛋白質，不但可造成癌症的發生；同時也具有引發免疫反應的能力，常用於腫瘤疫苗的研發。其中，又以E7蛋白的長效性表現與可引發較強免疫反應的緣故，最具有發展成子宮頸癌疫苗的價值。
李斯特菌為革蘭氏陽性，且絕對細胞內寄生的腸胃道細菌。它具有可躲避細胞內phagolysosome的能力。由於，李斯特菌行絕對細胞內寄生；所以感染後可引發很強的細胞性免疫反應。基於上述的特性；本實驗室利用基因重組方式，建構出一個帶有E7基因的重組型李斯特菌。利用口服方式給予帶有子宮頸癌的老鼠，證明在老鼠體內誘發出對E7有專一性的細胞毒殺反應。此一免疫反應對於腫瘤的消除與預防都有不錯的效果。更值得一提的是；口服疫苗本身除了給予方便、易被接受外，還有著降低生產成本的優點。故此一重組型李斯特菌，就功能上或給予上來說都是極具發展潛力的子宮頸癌腫瘤疫苗。

Cervical carcinoma remains one of the most common malignancies world wide with 500,000 new cases diagnosed each year, and 200,000 cervical cancer deaths annually. Human papillomavirus type 16 (HPV-16) is the high risk in casuse of cervical cancer and encodes three transforming oncogenes-E5, E6, and E7. They are thus unique tumor antigens and can be ideally used as tumor vaccines. Because E7 oncoprotein is consistently retained and expressed, and becomes more attractive target for T-cell-based immunotherapy of cervical cancer. Evidences for HPV antigen-directed immunotherapy against cervical cancer come from the experimental and natural papillomavirus-associated tumors that can be controlled by immunization with E7 antigen.
Listeria monocytogenes is a Gram-positive, facultative intracellular bacterium that has the unusual ability to escape from the phagolysosome. Live L. monocytogenes infection elicits a strong protective cellular immune response with the induction of both CD4 and CD8 T cells. In this study, we constructed the recombinant Listeria encoding HPV-16 E7 gene (rLM-E7). When challenge syngenic mice by oral recombinant rLM-E7, that could induce CTL response and tumorigenesis could be prevented and regressed via CD8-dependent CTL. Exceptly, oral vaccines are easier to administer, more acceptable to recipients, and less pure than the other vaccines formulated for injection, that reduces the cost of production.

正文目錄
標題 頁次
中文摘要 ------------------------------------------------ 1
英文摘要 ------------------------------------------------ 2
前言 ---------------------------------------------------- 3
人類乳突瘤病毒與子宮頸癌的關係 -------------------------- 3
人類乳突瘤病毒的基本特性與分型 -------------------------- 5
人類乳突瘤病毒的基本構造 -------------------------------- 6
目前子宮頸癌腫瘤疫苗策略 -------------------------------- 7
細菌性基因載體~李斯特菌(Listeria monocytogenes) --------- 8
利用重組李斯特菌發展成腫瘤疫苗的優點 -------------------- 9
材料與方法
建構能表現人類乳突瘤E7蛋白的重組型李斯特菌 ------------ 11
李斯特菌的培養 ------------------------------------------ 11
野生型與重組型李斯特菌的篩選 ---------------------------- 12
西方點墨分析法(Western blot) -------------------------- 12
細胞培養 ------------------------------------------------ 16
蛋白片段合成(Synthesis of related peptides) ------------ 18
C57BL/6J老鼠口服疫苗免疫反應分析 ------------------------ 18
細胞表面標記與細胞激素雙染色 ---------------------------- 19
腫瘤老鼠口服疫苗試驗動物模式 ---------------------------- 20
建立轉移性腫瘤老鼠模式 ---------------------------------- 21
結果
建構帶有HPV-16 E7基因的重組型李斯特菌 ------------------ 23
確認重組型李斯特菌可表現HPV-16 E7蛋白質 ---------------- 23
利用雙染色法(Double Stain Assay)分析重組型李斯特
菌所引發免疫反應的趨勢 ---------------------------------- 24
在老鼠體內，口服重組型李斯特菌對於能表現E7蛋
質的腫瘤細胞具有預防的效果 ------------------------------ 25
在老鼠體內，口服重組型李斯特菌對於能表現E7蛋
質的腫瘤細胞具有治療的效果 ------------------------------ 25
利用基因剔除老鼠模式，在活體中確定口服李斯特菌
可經由細胞性免疫反應的活化來對抗腫瘤細胞的生長 ----------- 26
在老鼠體內，口服重組型李斯特菌對於能表現 E7 蛋
白質的轉移性腫瘤是否具有清除的效果 --------------------- 27
評估口服重組型李斯特菌疫苗所引發不同免疫強度的
時間變化 ------------------------------------------------ 28
討論
以李斯特菌為載體來發展的口服性疫苗 ---------------------- 40
李斯特菌發展疫苗的優點與結果 ---------------------------- 40
李斯特菌發展疫苗的展望 ---------------------------------- 41
參考文獻 ------------------------------------------------ 43
圖表目錄
標題 頁次
圖一 -------------------------------------------------- 29
圖二 -------------------------------------------------- 30
圖三 -------------------------------------------------- 31
圖四 -------------------------------------------------- 32
圖五 -------------------------------------------------- 33
圖六 -------------------------------------------------- 34
圖七 -------------------------------------------------- 35
圖八 -------------------------------------------------- 36
圖九 -------------------------------------------------- 37
圖十 -------------------------------------------------- 38
圖十一 --- ---------------------------------------------- 39

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