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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/42438完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 陶秘華(Mi-Hua Tao) | |
| dc.contributor.author | Yo-Yu Tseng | en |
| dc.contributor.author | 曾友瑜 | zh_TW |
| dc.date.accessioned | 2021-06-15T01:13:47Z | - |
| dc.date.available | 2014-09-15 | |
| dc.date.copyright | 2009-09-15 | |
| dc.date.issued | 2009 | |
| dc.date.submitted | 2009-07-29 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/42438 | - |
| dc.description.abstract | 在慢性B型肝炎病人當中，通常病人無法經由免疫反應來清除體內的病毒。因此，如何有效地引發慢性B型肝炎病人體內的免疫反應來對抗受到HBV感染的肝臟細胞以達到清除HBV的策略正被積極的發展中。
治療性疫苗具有免疫調節的能力，並且幾乎沒有副作用。然而在帶有慢性B型肝炎病人中使用疫苗卻無法表現抗病毒的能力，引起的免疫反應也很低落。不過在病毒量降低的情況下，治療性疫苗不但可以引發免疫反應，並且其效果比起在病毒量較高的時候還要好。因此我們想要測試若是在帶有HBV的基因轉殖鼠上降低HBV病毒量之後再使用疫苗免疫，其疫苗效果是否比起未降低病毒量時還要來的有效。 在我們實驗室2007年發表的文獻指出使用雙股腺相關病毒-8作為載體(double-stranded adenoassociated virus 8-pseudotyped vector; dsAAV2/8)攜帶針對HBV的shRNA，發現帶有HBV-S1的shRNA之AAV(dsAAV2/8/HBV-S1)可以有效的長期抑制HBV基因轉殖鼠體內的hepatitis B surface antigen (HBsAg)以及HBV DNA量。除此之外，dsAAV2/8/HBV-S1也幾乎完全清除了基因轉殖鼠肝臟細胞中的hepatitis B core Ag (HBcAg)。 因此在降低病毒量之後，我們使用可以表現HBcAg的DNA疫苗以及腺病毒疫苗以DNA prime-adenovirus boost的策略免疫老鼠，發現可以有效地引起基因轉殖鼠體內的CD8+ T cell分泌IFN-γ。並且我們降低病毒量之後選擇了不同時間點進行免疫，發現超過8週之後再注射疫苗，就不容易引起CD8+ T cell分泌IFN-γ。 除了降低基因轉殖鼠體內的HBV病毒量可以增強疫苗的效果，我們也嘗試使用佐劑(adjuvant)來加強疫苗的效果。Granulocyte/monocyte colony-stimulating factor (GM-CSF)目前被公認為是促使樹突細胞(dendritic cells; DCs)成熟非常重要的一個細胞激素(cytokine)。CD40 ligand (CD40L)，主要表現在已活化T細胞表面的輔助刺激分子(costimulatory molecule)。因此我們使用可以製造出CD40L/GM-CSF融合蛋白的質體作為我們疫苗的佐劑。 我們發現若是以cardiotoxin預處理過後的老鼠，使用CD40L/GM-CSF融合蛋白的質體佐劑可以有效地加強疫苗引起的抗體反應，並且也可以增強T cell的增生能力。 總而言之，使用疫苗之前先降低病毒量是可值得繼續研究的方法。另外結合兩種佐劑成為一種融合性蛋白來加強疫苗效果，或許是個值得期待的研究方向。 | zh_TW |
| dc.description.abstract | In chronic hepatitis B virus infection, patients usually cannot eliminate virus by immune response themselves. Therefore, developing the stratrgy to trigger the immune response of chronic hepatitis B patients against HBV infected liver cells is actively persuaded.
Therapeutic vaccine exhibits potent immune modulatory potentials and almost no side effects, but possesses little antiviral capacity and immune response in patients with chronic hepatitis B. However, reducing virus titer can enhance therapeutic vaccine efficacy, and acquire better immune response than in higher virus titer condition as well. Therefore, we tried to test whether performing the therapeutic vaccine after reducing virus titer in HBV transgenic mice can get more efficient response than those mice didn’t reduce virus titer. In our previously study published in 2007, using double-stranded adeno-associated virus 8-pseudotyped vector (dsAAV2/8) cantaining shRNA specific to HBV, especially HBV-S1 (dsAAV2/8/HBV-S1), can long-term inhibit the hepatitis B surface antigen (HBsAg) and HBV DNA in HBV transgenic mice. And the inhibition can be lasting for 120 days. Besides, dsAAV2/8/HBV-S1 almost clears the hepatitis B core Ag (HBcAg) of hepatocyte in transgenic mice. After inhibiting virus titer in transgenic mice, we used DNA vaccine and adenovirus vaccine which encode HBcAg to immunize mice with DNA prime-adenovirus boost strategy. We found out this strategy can efficiently induce CD8+ T cell secreting IFN-γ. Meanwhile, we also choose several time points of immunization after HBV titer reduction. As vaccine administration was longer than 8 weeks after inhibiting virus titer, it showed no IFN-γ secreting response. Despite of reducing HBV titer in transgenic mice in order to enhance vaccine efficacy, we also tried to improve vaccine ability by using adjuvants. Granulocyte/monocyte colony-stimulating factor (GM-CSF) is now considered as an important cytokine to make dendritic cell maturation. CD40 ligand (CD40L) is a costimulatory molecule major expressing on activated T cell. Therefore, we construted the expression vector that encodes CD40L/GM-CSF fusion protein as our vaccine adjuvant. We found that using plasmid encoding CD40L/GM-CSF fusion protein in cardiotoxin pretreated mice can efficiently enforce vaccine-induced antibody response. Additionly, it can improve T cell proliferation. In conclusion, reducing virus titer before vaccination will be a strategy worth studying. Meanwhile, combination of two adjuvants as a fusion protein might be an inspiring method to improve vaccine efficacy. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T01:13:47Z (GMT). No. of bitstreams: 1 ntu-98-R96424012-1.pdf: 1206427 bytes, checksum: ca45285c60ace79556f827bdfbbae44b (MD5) Previous issue date: 2009 | en |
| dc.description.tableofcontents | 目錄
誌謝 i 摘要 ii Abstract iv 縮寫表 vi 目錄 vii 圖目錄 xi 第一章、 緒論 1 第一節、 B型肝炎病毒背景介紹 1 一、 發現歷史 1 二、 B型肝炎病毒病毒學 1 三、 急性與慢性B型肝炎免疫反應 2 第二節、 降低HBV病毒量以及使用疫苗引發免疫反應 2 一、 B型肝炎病毒藥物發展 2 二、 B型肝炎病毒疫苗發展 3 三、 結合藥物與疫苗作為B型肝炎病毒的治療策略 4 四、 降低病毒量提高疫苗功效 5 五、 使用shRNA抑制B型肝炎病毒量 5 六、 疫苗抗原的選擇 5 七、 在基因轉殖鼠上使用疫苗克服免疫耐受性 6 第三節、 發展搭配疫苗使用的佐劑 7 一、 佐劑背景介紹 7 二、 GM-CSF的生物功能 7 三、 GM-CSF作為疫苗佐劑 8 四、 CD40 ligand的生物功能 8 五、 可溶性三聚體CD40L 9 六、 CD40L作為疫苗佐劑克服T cell的週邊耐受性 9 七、 結合GM-CSF以及CD40L兩種分子作為疫苗佐劑 10 第四節、 論文研究動機與目的 10 一、 使用治療性疫苗克服基因轉殖鼠體內的免疫耐受性 10 二、 發展佐劑加強疫苗效果 10 第二章、 實驗材料和方法： 12 第一節、 質體的構築 12 一、 聚合酵素連鎖反應 12 二、 1%洋菜膠電泳 16 三、 DNA片段分離 16 四、 限制酵素切割反應 17 五、 DNA接合反應 17 六、 細菌熱休克轉殖法 17 七、 質體的製備 17 第二節、 細胞株培養與細胞表現檢驗 18 一、 細胞株 18 二、 真核細胞的轉染 19 三、 SDS-PAGE 21 四、 西方點墨法 22 五、 酵素連結免疫分析法 23 第三節、 實驗動物 25 第四節、 質體DNA免疫注射 25 第五節、 T cell分離方法 26 一、 磁珠分離法 26 二、 Nylon-wool法 28 第六節、 T cell分析實驗 30 一、 IFN-γ酶聯免疫斑點法 30 二、 T細胞增生試驗 32 第三章、 實驗結果 34 第一節、 構築疫苗質體 34 一、 p3224-3-HBV core 34 第二節、 確認疫苗質體所製造蛋白質 34 一、 免疫螢光染色法 34 第三節、 使用疫苗質體免疫老鼠 35 一、 測試p3224-3-HBV core質體引起免疫反應之能力 35 第四節、 降低病毒量之後使用疫苗 35 一、 觀察dsAAV2/8/shRNA在HBV/HLA-A*0201 (HBV/A2)基因轉殖鼠體內抑制HBV病毒量情形 35 二、 以dsAAV2/8/HBV-S1抑制HBV/HLA-A*0201 (HBV/A2)基因轉殖鼠體內HBV病毒量之後，測試疫苗免疫效果 36 三、 在dsAAV2/8/HBV-S1與dsAAV2/8/GL2兩種組別比較HBV core的疫苗效果 37 第五節、 不同時間點注射疫苗造成的影響 37 一、 在不同時間點進行疫苗免疫對於HBV病毒量的影響 37 二、 測試不同時間點進行疫苗免疫對於免疫反應的影響 38 第六節、 使用佐劑加強疫苗的效果 38 第七節、 構築佐劑質體 39 一、 p3224-3-GM-CSF 39 二、 p3224-3-IgκL-ILZ-CD40L 39 三、 p3224-3-IgκL-ILZ-CD40L/GM-CSF 40 第八節、 確認佐劑質體所製造蛋白質 40 一、 西方點墨法 40 二、 GM-CSF的生物功能 41 三、 CD40L的接合功能 41 第九節、 使用表現HBsAg疫苗質體搭配佐劑質體免疫老鼠 42 一、 免疫前先注射cardiotoxin，再使用pGM、pCD40L以及pCD40L/GM作為疫苗佐劑免疫C57BL/6老鼠 42 二、 免疫前先注射cardiotoxin，再使用pGM、pCD40L以及pCD40L/GM質體作為疫苗佐劑免疫B10.M老鼠 43 三、 疫苗搭配佐劑免疫過後的B10.M老鼠分析其體內T cell的增生能力 44 第四章、 討論 46 第一節、 表現HBcAg的質體引起的免疫反應 46 第二節、 有無抑制HBV病毒量對於疫苗所引起免疫反應的影響 47 第三節、 HBV/HLA-A*0201以及HLA- HLA-A*0201兩種基因轉殖鼠免疫反應比較 48 第四節、 施打疫苗時間點的選擇 48 第五節、 利用帶有GM-CSF以及CD40L的質體作為佐劑 49 第六節、 比較C57BL/6以及B10.M老鼠抗體反應差異 51 第七節、 採用cardiotoxin捨棄electroporation的原因 52 第八節、 使用佐劑加強治療性疫苗的效果 52 第五章、 參考文獻 54 圖目錄 圖 一、構築p3224-3-HBV-core質體 60 圖 二、HBcAg基因序列 61 圖 三、質體p3224-3-HBV-core表現HBcAg 62 圖 四、測試表現HBcAg的疫苗且以DNA prime-adenovirus boost免疫策略進行免疫的效果 63 圖 五、dsAAV2/8/shRNA在不同時間點對HBV/HLA-A*0201基因轉殖鼠所造成的影響 64 圖 六、經由dsAAV2/8/HBV-S1治療過後的HBV/HLA-A*0201基因轉殖鼠對疫苗的免疫反應 65 圖 七、比較具有抑制HBV病毒量以及不具有抑制HBV病毒量之dsAAV2/8/shRNA治療過的老鼠對於疫苗的免疫反應 66 圖 八、注射dsAAV2/8/HBV-S1之後在不同時間點進行疫苗免疫對HBV/HLA-A*0201基因轉殖鼠體內HBV病毒量的影響 67 圖 九、選擇抑制HBV病毒量之後施打HBcAg疫苗最佳時間點 68 圖 十、GM-CSF與CD40L在活化淋巴球中所扮演的角色 69 圖 十一、構築p3224-3-GM-CSF 70 圖 十二、GM-CSF基因序列 71 圖 十三、構築質體p3224-3-IgκL-ILZ-CD40L/GM-CSF 73 圖 十四、IgκL-ILZ-CD40L/GM-CSF基因序列 74 圖 十五、構築質體p3224-3-IgκL-ILZ-CD40L 75 圖 十六、IgκL-ILZ-CD40L基因序列 76 圖 十七、佐劑質體的蛋白質表現 77 圖 十八、測試質體製造GM-CSF以及CD40L/GM-CSF融合性蛋白的生物功能 78 圖 十九、CD40L分子以及CD40L/GM-CSF融合性蛋白對於CD40的黏合能力 79 圖 二十、C57BL/6老鼠(H-2b)經由HBsAg疫苗搭配不同佐劑免疫後其體內anti-HBsAg抗體反應差異 80 圖 二十一、B10.M老鼠(H-2f)經由HBsAg疫苗搭配不同佐劑免疫後其體內anti-HBsAg抗體反應差異 81 圖 二十二、免疫過後的B10.M老鼠其T cell增生能力在不同佐劑之間的差異 82 | |
| dc.language.iso | zh-TW | |
| dc.title | 改進慢性B型肝炎治療性疫苗的策略 | zh_TW |
| dc.title | Strategies to Improve Efficacy of Therapeutic Vaccines for Chronic Hepatitis B Infection | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 97-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 李君男(Chun-Nan Lee),楊雅倩(Ya-Chien Yang),莊雅惠(Ya-Hui Chuang) | |
| dc.subject.keyword | B型肝炎病毒,治療性疫苗,shRNA,佐劑,顆粒/單核球群落刺激生長因子,CD40L,融合性蛋白, | zh_TW |
| dc.subject.keyword | Hepatitis B virus,therapeutic vaccine,shRNA,adjuvant,granulocyte/monocyte colony-stimulating factor (GM-CSF),CD40 ligand (CD40L),fusion protein, | en |
| dc.relation.page | 82 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2009-07-29 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 醫學檢驗暨生物技術學研究所 | zh_TW |
| 顯示於系所單位： | 醫學檢驗暨生物技術學系 | |
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