臺灣博碩士論文加值系統

樹突狀細胞是目前在人體內，被認定功能最強大的抗原呈現細胞，其可從造血幹細胞分化而來，因此本實驗是利用了體外無血清增殖造血幹細胞的技術，來做為大量生產樹突狀細胞的來源，並進一步找尋樹突狀細胞的誘導培養基配方與細胞激素最適化的組成。
根據文獻中的探討，並經由實驗測試，得知SCF、Flt-3 ligand、IL-1β、GM-CSF和TNF-α等五種細胞激素對於樹突狀細胞的誘導與分化是有正向幫助的，而後再利用陡升路徑法，找出其最適化的濃度。
除了細胞激素的找尋之外，同時還測試了不同種類的基礎培養基，如IMDM、RPMI-1640、M-199、α-MEM、H3000、DMEM/F12 與 DMEM，再配合已實驗測試出的細胞激素最適化濃度組成，最後可建立樹突狀細胞誘導與分化的最適化誘導培養基。
在建立最適化誘導培養基之後，進一步再檢測樹突狀細胞的特性，如經脂多醣刺激成熟前後之表面抗原測試分析 (CD1a、CD11c、CD14、CD40、CD80、CD83、CD86、HLA-DR、CD45 和 CD34)，利用Mixed Lymphocyte Reaction (MLR) 來檢測其誘導CD3+ T細胞增生的功能性，檢測吞噬能力與特定細胞激素的分泌能力等。
希望藉此研究建立能大量量產及功能性樹突狀細胞的誘導培養系統，並以此建立本實驗室未來對於樹突狀細胞後續研究的根基。

Dendritic cells (DCs) are the most powerful antigen presenting cells (APCs) and play a pivotal role in initiating the immune response, which differentiated from CD133+ hematopoietic stem cells (HSCs). Hence, we used the ex vivo expanded of hematopoietic stem cells as a source of DCs, and developed the optimal DCs induction medium.
In the previous study, we had developed a serum-free hematopoietic stem cells expansion system (SF-HSC medium), HSCs could expand in SF-HSC medium reaching 30-fold within one week.
According to the past researches, several cytokines, especially SCF, Flt-3 ligand, IL-1β, GM-CSF and TNF-α, have been identified as essential factors to induce and differentiate HSCs into DCs. Moreover, we tested the basal media (IMDM, RPMI-1640, M-199, α-MEM, H3000, DMEM/F12 and DMEM) combined with the various concentration of cytokines to finalize the optimal DC induction medium.
Finally, we confirmed the function and maturation of DCs by the assays of the mixed lymphocyte reaction (MLR), the ability of endocytosis, specific cytokines of secretion and the stimulation by lipopolysaccharides. When DCs become mature (mDCs), the specific surface markers of mDCs would change (CD1a, CD11c, CD14, CD40, CD80, CD83, CD86, HLA-DR, CD45 and CD34), the ability of endocytosis would be more complete and the ability of stimulation would increase when co-cultured with CD3+ T cells.
These results showed that DCs derived from the serum-free expanded CD133+ HSCs exhibited both characteristics and functions of DCs. Therefore, we believed that combination of HSCs serum-free expansion medium and DCs induction medium would generate large amounts of functional DCs and would be a promising cell source for the basic research and translation media in the near future.

摘要 …………………………………………………………………………… I
Abstract ……………………………………………………………………… III
目錄 ………………………………………………………………………… V
表目錄 ……………………………………………………………………… VII
圖目錄 …………………………………………………………………… VIII
第一章 緒論 ………………………………………………………………… 1
1.1 楔子………………………………………………………………… 1
1.2 研究動機與目的 …………………………………………………… 1
1.3 研究架構 …………………………………………………………… 2
第二章 幹細胞與樹突細胞之發展與介紹 ………………………………… 5
2.1 幹細胞 (stem cell) 簡介 ………………………………………… 5
2.1.1 幹細胞定義 …………………………………………………… 5
2.1.2 幹細胞的分類 ………………………………………………… 6
2.1.3 幹細胞的來源 ………………………………………………… 8
2.2 造血幹細胞 (hematopoietic stem cells, HSCs) 簡介 …………… 10
2.2.1 造血幹細胞與造血系統 ……………………………………… 10
2.2.2 造血幹細胞的鑑定 …………………………………………… 12
2.2.3 造血幹細胞的應用 ………………………………………… 14
2.3 樹突狀細胞 (dendritic cells, DCs) 簡介 ……………………… 14
2.3.1 樹突狀細胞的型態與生理位置 ……………………………… 14
2.3.2 樹突狀細胞的鑑定 …………………………………………… 15
2.3.3 主要機制 …………………………………………………… 16
2.3.4 目前遇到的問題 …………………………………………… 17
第三章 實驗材料與方法 …………………………………………………… 19
3.1 實驗儀器 ………………………………………………………… 19
3.2 實驗藥品與材料 ………………………………………………… 19
3.3 實驗步驟 ………………………………………………………… 23
3.3.1 從臍帶血中分離出CD133+的造血幹細胞 ………………… 24
3.3.2 細胞培養 (cell culture) ……………………………………… 25
3.3.3 增值後的造血幹細胞的初步誘導成樹突細胞 ……………… 26
3.3.4 細胞冷凍保存 ………………………………………………… 26
3.3.5 細胞激素 (cytokine) 最適化篩選 ………………………… 27
3.3.6 流式細胞儀分析 ……………………………………………… 30
3.3.7 樹突狀細胞成熟的刺激 ……………………………………… 32
3.3.8 吞噬能力 (Endocytosis) 分析 ……………………………… 32
3.3.9 細胞激素分泌 ………………………………………………… 33
3.3.10 T細胞刺激生長 (Mixed Lymphocyte Reaction, MLR) 33
第四章 實驗結果與討論 …………………………………………………… 35
4.1 樹突狀細胞誘導的可行性分析 ………………………………… 35
4.2 細胞激素之因子試驗篩選結果與分析 ………………………… 38
4.2.1 初篩因子試驗分析 ………………………………………… 38
4.2.2 複篩因子試驗分析 ………………………………………… 44
4.3 藉陡升路徑法找最適化細胞激素組成濃度 ………………… 51
4.3.1 陡升路徑之樹突狀細胞誘導結果 ……………………… 52
4.4 基礎培養基 (basal media) 之比較 …………………………… 59
4.4.1 基礎培養基的選擇 ………………………………………… 59
4.4.2 樹突狀細胞之誘導結果 …………………………………… 60
4.5 樹突狀細胞表面抗原分析 …………………………………… 65
4.5.1 樹突狀細胞表面抗原比例分析結果 …………………… 65
4.5.2 成熟樹突狀細胞表面抗原變化 …………………………… 72
4.6 吞噬能力 (Endocytosis) 分析 ………………………………… 79
4.6.1 吞噬能力的結果與數量表現 ……………………………… 80
4.7 細胞激素分泌量測試 …………………………………………… 82
4.7.1 細胞激素濃度分析結果 …………………………………… 82
4.8 T細胞刺激生長實驗 (MLR) ………………………………… 84
4.8.1 共同培養結果分析 ………………………………………… 85
第五章 結論與未來展望 …………………………………………………… 88
5.1 結論 ……………………………………………………………… 88
5.2 未來展望 ………………………………………………………… 90
參考文獻 …………………………………………………………………… 91

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