臺灣博碩士論文加值系統

主題一
在台灣，HLA (Human Leukocyte Antigen) -A24的族群佔了有60.4%，但是近年來有關於人類HLA的研究，都是著重於A2的族群，這是因為HLA-A2佔世界人口的比例是最高的。因此，本實驗主要的目標，就是建立一個可以研究HLA-A24胜肽疫苗的系統。利用重組慢病毒轉導T2細胞，成功的建立出T2/A24/10、T2/A24/18、T2/A24/25、T2/A24/30、T2/A24/42、T2/A24/54這六株能夠表現HLA-A24分子的抗原呈獻細胞，可用來篩選電腦軟體所預測的CTL(Cytotoxic T Lymphocyte) epitopes，評估是否可作為胜肽疫苗的選擇，之後再以A24的基因轉殖鼠進一步確認，這樣的CTL epitope是否可以在老鼠體能成功的引發針對此段epitope的免疫反應。
總而言之，這樣的一個研究HLA-A24胜肽疫苗的系統，可以作為將來研究腫瘤生長與疫苗學等研究的一個重要的工具。

主題二
子宮頸癌是世界女性癌症死亡率第二高的一種癌症，每年有約288,000人死於子宮頸癌。在之前的研究中，子宮頸癌的生成有很多因素，像是性荷爾蒙、抗細胞凋亡因子、上皮生長因子、血管上皮生長因子等等。近年來，單一核酸之多型性(Singlenucleotide polymorphism；SNP)及類固醇荷爾蒙受體和癌症的發生之間的關係，漸漸被證實。基因的單一核酸突變，例如，CYP1A1第3801個核甘酸的變異與肺癌的生成的關係。而類固醇荷爾蒙可以召集一些活化型輔因子獲抑制型輔因子來調控基因的表現。因此，本實驗室利用酵母菌雜交系統實驗，發現了一個新穎的細胞核受體交互作用蛋白(Nuclear receptor interaction protein；NRIP)，並且證實了NRIP和子宮頸癌的關係。之前的實驗證實了，NRIP會和雄性激素受體(Androgen receptor；AR)及醣皮質激素皮質醇受體(Glucocorticoid receptor；GR)結合，促進AR及GR所調控的基因表現。因此，NRIP與癌症之間詳細的關係還有待研究。
在本實驗中，我們研究了NRIP在正常組織及子宮頸癌組織中DNA序列的多型性，發現無論是在子宮頸癌組織或是正常組織，NRIP DNA的序列都沒有任何差異。除此之外，隋著轉錄因子YY1量的提高，NRIP啟動子的活性有些微上升的趨勢，這可能代表了YY1在這裡作為一個活化型因子。

Subject I
Differences in HLA gene have been observed between various ethnic groups of the Chinese population in Taiwan. High frequency of HLA-A24 (60.4%) was noticed in Taiwan inhabitants. But recent studies were aimed at HLA-A2 population since it’s the largest and most diverse allele family at the HLA-A locus. Therefore, generating a system for peptide vaccine research in HLA-A24 population is our primary goal. In this study, HLA-A24 Antigen-Presenting Cell was generated by lentivirus transduction(T2/A24/10, T2/A24/18, T2/A24/25, T2/A24/30, T2/A24/42, T2/A24/54). The HLA-A24 antigen presenting cell could provide a useful tool for screening of computer predicted CTL epitopes. In conclusion, this HLA-A24 CTL epitopes screening system, HLA-A24 antigen presenting cell and HLA-A24 Tg mice, could be applied in many fields, the development of tumor and molecular biology, vaccinology, immunology and cancer research.

Subject II
Cervical cancer is the second biggest cause of female cancer mortality worldwide with 288,000 deaths yearly. Following previous reports, many other factors were involved in tumorgenesis of cervical cancer, like sexual hormone, anti-apoptosis factor, EGF and VEGF. In recent years, the roles of steroid and steroid hormone receptor in the occurrence of cancer are widely recognized. In the past, it was demonstrated that steroid hormone receptors can modulate the gene expression by recruiting co-activators or co-repressor to target gene promoter. In this way, we found a novel transcription co-activator, NRIP, by yeast-two-hybrid system and its association with cervical cancer was also explored. In our study, the NRIP was found to interact with AR or GR by in vitro co-immunopreciptation assay and could enhance AR- or GR- mediated transcription activity. Therefore, the role which NRIP plays in cancer is under investigation.
In this study, DNA sequence variations of NRIP between cervical cancer and normal tissue were evaluated and no SNP was fouond. Besides, the regulatory mechanism of NRIP promoter activities was also measured by luciferase assay. These results indicate that YY1 could play an activation role in NRIP promoter activities in a dose-dependent manner.

正文目錄 Ⅰ
圖表目錄 Ⅴ
中文摘要 Ⅵ
Abstract Ⅷ
第一篇 建立T2/A24抗原呈現細胞作為CTL胜肽片段之篩選工具 1
第一章 緒論 1
第一節 胜肽疫苗(Peptide-based vaccine) 1
第二節 T2細胞 3
第三節 T2細胞結合試驗 4
第四節 台灣HLA(Human Leukocyte Antigen)分布 4
第五節 研究策略. 5
第二章 材料及方法 6
第一節 細胞培養 6
壹 293T細胞 6
貳 T2細胞 6
參 TE671細胞 7
第二節 重組慢病毒(Lentivirus)載體之構築 7
壹 引子(Pimer)設計 8
貳 重組慢病毒載體之構築 8
一 Insert DNA (HLA-A24 cDNA) preparation 8
二 Vector DNA (pTY-EF1α vector) preparation 8
三 Ligation of pTY-EF1α and A24 fragment 9
四 Transformation 9
第三節 pTY-EF1α/A24載體在真核細胞中之表現 9
壹 293T細胞之轉染. 10
貳 流式細胞儀分析 10
第四節 A24重組慢病毒之製備 10
壹 293T細胞之轉染 11
貳 重組慢病毒之濃縮 11
参 重組慢病毒之效價測定 11
第五節 建立表現A24分子之T2細胞株 12
壹 A24重組慢病毒轉導(transduction)T2細胞 12
貳 96孔盤分離Single clone 12
参 細胞核去氧核醣核酸(genomic DNA)之萃取 13
肆 PCR篩選 13
第六節 建立表現A24分子之T2細胞株 13
壹 引子設計 14
貳 細胞Total RNA萃取 14
参 Genomic DNA removal 15
肆 RT-PCR(反轉錄聚合酶鏈反應) 15
第三章 結果 16
第一節 重組慢病毒(Lentivirus)載體之構築 16
第二節 pTY-EF1α/A24質體在真核細胞中之表現 16
第三節 A24重組慢病毒之製備與效價測定 16
第四節 重組慢病毒之劑量選擇 17
第五節 建立T2/A24細胞株 17
第四章 討論 19
第五章 結論 23
第六章 參考文獻 24

第二篇 評估新穎核受體交互作用蛋白, NRIP, 在子宮頸癌之作用 36
第一章 緒論 36
第一節 新穎細胞核受體交互作用蛋白(NRIP) 36
第二節 單一核酸之多型性(Singlenucleotide polymorphism；SNP) 37
第三節 調節輔因子(Co-regulators)與癌症 37
第四節 研究策略 38
第二章 材料及方法 39
第一節 細胞培養 39
壹 293T細胞 39
貳 CaSki細胞 39
第二節 比較NRIP在人類正常及子宮頸癌細胞基因層次之變化 40
壹 細胞核去氧核醣核酸(Genomic DNA)之萃取 40
貳 外顯子(Exon)引子設計 40
第三節 比較NRIP啟動子在人類正常及子宮頸癌細胞之差異 44
壹 NRIP啟動子引子設計 44
第四節 探討YY1對NRIP啟動子之作用 45
壹 構築剔除YY1結合區域之NRIP啟動子質體 45
一 引子設計 45
二 Insert DNA (dYY1NRIP fragment) preparation 46
三 Vector DNA (pGL3Basic vector) preparation 46
四 Ligation of pGL3Basic and dYY1NRIP fragment 46
五 Transformation 46
貳 YY1表現載體之構築 47
一 Insert DNA (YY1 fragment) preparation 47
二 Vector DNA (pcDNA3.1/HisC vector) preparation 47
三 Ligation of pcDNA3.1/HisC and YY1 fragment 47
四 Transformation 48
参 細胞轉染 48
一 剔除YY1結合區域對NRIP啟動子活性之影響 48
二 YY1蛋白對NRIP啟動子之作用 49
肆 細胞全蛋白(Total protein)萃取 49
伍 蛋白質之定量 50
陸 螢火蟲冷光活性法(Luciferase assay) 50
一 Luciferase detection 50
二 β-Galactosidase detection 50
三 Luciferase value normalization 50
第三章 結果 51
第一節 比較NRIP在人類正常及子宮頸癌細胞基因層次之變化 51
第二節 比較NRIP Promoter在人類正常及子宮頸癌細胞之差異 52
第三節 探討YY1對NRIP啟動子之作用 53
第四章 討論 55
第五章 結論 57
第六章 參考文獻 58
附錄 75

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