臺灣博碩士論文加值系統

在過去的研究中，我們實驗室以人類週邊單核細胞 (Human peripheral blood mononuclear cells; PBMC) 為標的細胞 (Target cells)，進行具有免疫調控活性之天然物篩選，發現由蘭嶼肉桂 (Cinnamomum kotoense)、牛蒡子 (Arctium lappa) 及瓊麻 (Agave sisalana) 純化出的天然物，分別為kaempferol 3-O-a-L-[2,4-di-(E)-p-coumaroyl]rhamnopyranoside (K3)，arctigenin (AC) 及 3,9-dihydroeucomin (DC)，可有效抑制由植物凝集素 (Phytohemagglutinin; PHA) 引起之 PBMC 增生現象。由於 PHA 刺激之主要細胞為 T 淋巴細胞，為了瞭解這些天然物是否真正作用於 PBMC 中的 T 淋巴細胞，並瞭解其部分作用機轉，研究中以Anti-CD3 與 Anti-CD28 抗體刺激純化之人類T 淋巴細胞活化為模式，探討K3、 AC 與 DC 對於人類T 淋巴細胞免疫反應之調控作用。實驗結果顯示：(1)利用 Nylon wool 分離法，由人類 PBMC 中純化 T 淋巴細胞，以 Anti-CD3 抗體進行免疫螢光染色，利用流式細胞儀 (Flow cytometer) 鑑定T 淋巴細胞純度達80% ；(2)以反轉錄聚合酶連鎖反應(Reverse Transcription-Polymerase Chain Reaction; RT-PCR) 分析 Anti-CD3 與 Anti-CD28抗體所引起 T 淋巴細胞中 IL-2 及 IFN-r基因表現於2小時最為明顯；(3)利用酵素連結免疫測定法 (ELISA) 檢測細胞於 Anti-CD3 與 Anti-CD28 antibodies 刺激 24 小時情況下，可偵測到 IL-2 及 IFN-r 蛋白質產生；(4)利用 3H-Thymidine uptake 檢測方法，發現以 Anti-CD3 與 Anti-CD28 抗體刺激48小時，可明顯造成 T 淋巴細胞活化增生現象；(5)以 RT-PCR 方法檢測出K3、 AC 與 DC，能降低Anti-CD3 與 Anti-CD28 抗體所引起 T 淋巴細胞中 IL-2 及 IFN-r之基因表現；(6)以 ELISA 方法檢測細胞上清液，發現K3、 AC 與 DC能降低由Anti-CD3 與 Anti-CD28 抗體所引起之 T 淋巴細胞中 IL-2 及 IFN-r蛋白質產生；(7)利用 3H-Thymidine uptake 方法，發現K3、 AC 與 DC能顯著抑制由 Anti-CD3 與 Anti-CD28 抗體所引起之 T 淋巴細胞活化增生現象；(8)以 Alamar blue 方法鑑定出K3、 AC 與 DC對於 T 淋巴細胞無直接細胞毒性作用；(9)在Anti-CD3 與 Anti-CD28 抗體刺激情況下，利用各種抑制劑加入，發現 ERK、 P38、 NF-kB 與 NF-AT 等訊息因子活化，參與了 IL-2 與 IFN-r基因表現之調控途徑；(10) Western blotting 結果顯示K3、DC與AC 對於 ERK 之活化不具抑制活性，但 DC 對於 P38 之活化具有些微之抑制活性；(11) Luciferase Reporter Assay 初步結果發現， K3 能減少 NF-AT 轉錄因子 (Transcription factor) 的活化; (12) EMSA結果顯示 K3 確實能減少 NF-AT、NF-kB轉錄因子的活化，而 AC 與 DC 對其則無抑制作用。綜合以上研究結果顯示，蘭嶼肉桂、牛蒡子及瓊麻中含有免疫調控因子 (Immunomodulatory factors)，它們在不直接毒殺細胞情況下，能有效降低 IL-2 與 IFN-rmRNA 表現與蛋白質產生，進而抑制人類 T 淋細胞活化增生現象，其中 K3 可能藉由抑制NF-AT、NF-B轉錄因子活化而調控 T 淋巴細胞免疫反應。藉由此些研究數據建立，希望未來可做為由中草藥開發免疫調控劑之依據。

The immune system is a collection of mechanisms within an organism that protects against disease by identifying and killing pathogens and tumor cells. However, overactive effector immune responses to autologous or allogeneic antigens can result in immune-mediated diseases such as hypersensitivity and autoimmune diseases. One of the therapeutic objectives in autoimmune diseases is blocking of activation, proliferation and cytokine production in T lymphocytes. There are immunosuppressive drugs used for autoimmune diseases, but they almost accompany with serious side effects. Therefore, we attempted to identify other immunomodulators from natural products that could be as an adjuvant treatment for autoimmune diseases. The previous data indicated that the natural products kaempferol 3-O-a-L-[2,4-di-(E)-p-coumaroyl] rhamnopyranoside (C39H32O14; MW=724; K3), arctigenin (C21H24O6; MW=372; AC), and (±)-3,9-dihydroeucomin (C17H16O5; MW=300; DC) isolated from Cinnamomum kotoense, Arctium lappa and Agave sisalana, respectively, inhibited human peripheral blood mononuclear cells (PBMC) proliferation stimulated by phytohemagglutinin (PHA). In the present study, human T lymphocytes were purified from PBMC and used as target cells. The regulation of K3, AC, and DC in human T lymphocytes immune responses induced by anti-CD3 and anti-CD28 antibodies (anti-CD3/CD28 Abs) was studied. The results indicated that (1) Primary human T lymphocytes has been isolated from PBMC by nylon wool method and their purities are about 80%. (2) Both IL-2 and IFN-r mRNA expression and protein production could be detected in T lymphocytes induced by anti-CD3/CD28 Abs at 2 hr and 24 hr postactivtion, respectively. The cell proliferation in T lymphocytes could be detected at 72 hr postactivation. (4) Both IL-2 and IFN-r gene expression in T lymphocytes activated anti-CD3/CD28 Abs were regulated by ERK, P38, NF-kB, and NF-AT signaling pathways. (5) All K3, AC and DC reduced IL-2 and IFN-rmRNA transcripts and protein production and cell proliferation in T cells induced by anti-CD3 and anti-CD28 antibodies without cytotoxicity. (6) Both K3 and AC did not affect ERK and P38 activation in T lymphocytes stimulated with anti-CD3/CD28 Abs. DC decreased P38 phosphorylation in activated T lymphocytes. (7) The preliminary data from luciferase reporter assay showed that K3 and AC decreased NF-AT activation. (8) The results from electrophoresis mobility shift assay (EMSA) demonstrated that K3 impaired NF-AT and NF-kB DNA binding activities. We suggested that K3 suppressed IL-2 and IFN-r gene expression in T lymphocytes induced by anti-CD3/CD28 Abs related to reduction of NF-AT and NF-B activity. Furthermore, the detailed action mechanisms behind the differential effects of K3, AC, and DC on human T lymphocytes will be elucidated.

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中文摘要 ------------------------------------ 5

英文摘要 ------------------------------------ 6

壹、緒言 ------------------------------------ 7

貳、材料與方法 ------------------------------ 19

参、結果 ------------------------------------ 31

肆、討論 ------------------------------------ 45

伍、參考文獻 -------------------------------- 54

附錄 ---------------------------------------- 60

表 ------------------------------------------ 61

圖 ------------------------------------------ 63

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