2012-08-012024-05-15https://scholars.lib.ntu.edu.tw/handle/123456789/664212摘要：抗原遞輸在疫苗發展中扮演極重要之角色。疫苗通常須要特殊的遞送系統以加強其免疫反應。然而，大部分利用疫苗佐劑以促進免疫反應之免疫機制仍不清楚。在此計畫中，我們提出一個假設，即會造成cross-priming之疫苗佐劑會促進血液細胞之分化，尤其是針對血液幹細胞。為測試此一假設，我們擬用一些常用之疫苗佐劑，包括alum、complete Freund'sadjuvant、 incomplete Freund's adjuvant、與含pluronic L121之乳劑疫苗佐劑等。這些疫苗佐劑將與雞卵蛋白以皮下注射方式打入C57BL/6 小鼠。經boosting後，將以皮下注射方式(s.c.)將含ovalbumin (OVA)疫苗佐劑 (Alum, complete Freund’s adjuvant, L121-adjuvant等) 打入小鼠體內使其產生免疫反應，每隔七天boost一次。然後每隔一段時間由尾靜脈採血，以ELISA分析小鼠體內測其抗體與cytokines變化情形。小鼠犧牲後取大腿骨及小腿骨，抽取骨髓細胞，與其他免疫器官細胞 (包括胸腺、脾臟、與腹側淋巴結等)，離心後以螢光抗體染色，再用流式細胞儀分析，並分析其intracellular cytokine expression.等實驗。在有關adoptive transfer實驗方面，擬將取C57BL/6J mice小鼠之血液前驅細胞(progenitor cells)，探討其在經各種疫苗佐劑處理後之CD45.1小鼠體內所產生分化與免疫反應之能力。將獲取C57BL/6J mice小鼠之骨髓細胞，再以流式細胞儀篩選 lineage- c-kit+sca-1+(LSK)、CLP (common lymphoid progenitors)、與CMP (common myeloid progenitors)等細胞。再將sorted cells 打入CD45.1小鼠骨髓腔。經數星期後，將小鼠犧牲後，取骨髓與脾臟細胞，然後以流式細胞儀分析lineage, CD11b, Ly-6G, Gr-1, CD115, MHC II等之表現。有關Gr-1+/CD11b+ 調控細胞刺激或抑制 T cells proliferation之實驗則擬將取經疫苗佐劑處理後之C57BL/6J 小鼠骨髓細胞，以流式細胞儀篩選所要細胞群(Gr-1+/CD11b+) 。再將OT-1小鼠CD8+脾臟細胞經negative selection純化後，以carboxyfluorescein succinimidyl ester (CFSE)-染色，然後與sorted Gr-1+/CD11b+細胞共培養數日後，以流式細胞儀分析CD8+ T cells之proliferation。同時dendritic cells 之precursors包括CDP 與MDP 亦相同方式以檢視疫苗佐劑對於數突細胞之影響。此計畫之實驗結果，將有助於我們對於會產生cross priming之疫苗佐劑，其對於血液細胞分化之影響。<br> Abstract: Antigen delivery plays important roles in the development of vaccines. Despite the extensiveuse of vaccine adjuvants in antigen delivery, their roles in the enhancement of immunogenicityremain unclear. In this proposal, we hypothesize that the vaccine adjuvants for cross priminginduce the differentiation of hematopoietic system, resulting in modification of the development ofhaematopoietic stem cells. (HSC) To test this hypothesis, commonly used vaccine adjuvants,including alum, complete Freund's adjuvant, incomplete Freund's adjuvant, and L121-adjuvant,containing Pluronic L121, a tri-block copolymer, will be employed in this study. These adjuvantscontaining the ovalbumin (OVA) antigens will be injected s.c. into the C57BL/6 mice. Theanimals will be sacrificed after boosting, cells will be isolated from both the primary and secondarylymphoid/myeloid organs, such as bone marrow, spleen, and lymph nodes, followed by flowcytometric analysis of the expression of surface phenotypes such as lineage, CD11b, Ly-6G, Gr-1,CD115, MHC II. Thymus of the animals will also be collected for examination of the expressionof CD4, CD8, CD25, CD44, c-kit, and lineage etc.. To examine the lineage-specificdifferentiation of hematopoiesis as modified by the vaccine adjuvants, the hematopoieticprogenitors, mainly HSC (lineage-/Sca-1 +/c-kit+) cells, will be isolated from CD45.2 mice andintra-bone-marrow transferred into CD45.1 mice. The animals will be sacrificed after boostingand analyzed for the myeloid and lymphoid. Similarly, CLP (common lymphoid progenitors) andCMP (common myeloid progenitors) will also be isolated, followed by adoptive transfer andanalyzed for the development of myeloid lineages. To examine the cell population involved inantigen-specific immune response stimulated by the vaccine adjuvants, lineage-/Gr-1+/ CD11b+cell populations will be isolated from bone marrow after vaccination, and co-culture with5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled CD8+ T cells from the spleensof OT-1 mice, followed by flow cytometric analysis for T cells proliferation. The experimentalresults obtained from this study will clarify the roles of vaccine adjuvants in modification ofdifferentiation potential of the hematopoietic system.疫苗佐劑抗原傳輸血液細胞vaccine adjuvantantigen deliveryhematopoietic cells