點帶石斑魚(Epinephelus coioides) 為台灣重要的高經濟價值養殖魚類，然而近年來隨著高密度的養殖，併發了許多問題及疾病產生，導致養殖魚業重大的經濟損失，為了能有效預防及控制魚病的爆發，有必要對其免疫系統進行研究。Interleukin 6 (IL-6) 為在溝通及調節免疫反應的重要細胞激素之一，主要由T cells及巨噬細胞所分泌，具有可促使T cells及B cells增生及分化並可促進B cells產生抗體。本研究首次由點帶石斑魚中取得IL-6基因序列，其可轉譯出223個胺基酸，其胺基酸序列與哺乳動物相似度約30%，與其他已知魚種相比只有43%到63%，預測之蛋白結構由信號序列及4個保留α-螺旋結構所組成，則與哺乳動物以及其他魚種之IL-6近似。在il-6基因的研究中，在離體培養的週邊血液白血球中以LPS刺激後8小時會有大量表現的現象。在魚隻中鰓、頭腎、腦及肌肉有持續性的il-6表現，經過LPS的刺激24小時後，則在所有的免疫組織的表現量都會有明顯的上升；另外，以本研究所製備抗IL-6多株抗體以進行組織免疫染色，結果顯示在LPS刺激後8小時，在頭腎組織中IL-6陽性反應細胞數量達到最高量。在點帶石斑魚IL-6體內生物功能的研究上。我們利用大腸桿菌生產重組的石斑魚IL-6，經由腹腔注射方式刺激魚體後偵測各時間各免疫相關基因之表現量來進行功能性的探討。結果顯示，在發炎前細胞激素如tnf-α1、tnf-α2、il-1β及Th1專一性轉錄因子t-bet在IL-6刺激後有表現量下降的趨勢，而Th2專一性轉錄因子c-maf 和gata以及Ig則表現量有明顯上升，此外也觀察到急性期反應蛋白crp在肝臟也有大量表現的現象。根據目前的結果IL-6確實在魚類免疫系統中扮演重要角色且具調控助手T細胞走向分化Th2細胞的功能，而這個轉變則有助於抗體的產生。

Epinephelus coioides (orange-spotted grouper) is an economic marine fish of aquaculture in Asia. But the intensive rearing increases the susceptibility of infection and disease, which can cause heavy loss. Therefore, in order to control disease, it is essential to understand the immune system background of the grouper. The cytokines, interleukin 6 (IL-6), produced by T cell and macrophages plays an important role in immune response. It regulates the proliferation and differentiation of T cells and elicits immunoglobulin produced by B cells. In this study, we try to figure the biofunction of grouper IL-6. First, we obtain the il-6 of grouper. The deduced grouper IL-6 protein was a 223 amino acid protein and the amino acid sequence is about 30% identical to mammalian IL-6 and 43% to 63% identical to other available teleost IL-6. The putative conformation was composed by a signal peptide and four conserved alpha helices, which are the same as their counterparts of mammalian and teleost. In the mRNA expression pattern, peripheral blood leukocytes had a high expression of il-6 at 8 hours after LPS stimulation in the in vitro condition. In the in vivo condition, il-6 was continuously expressed in gill, head kidney, brain and muscle without LPS stimulation; however, il-6 was expressed in all immune organ in the fish injected with LPS. In the in vitro condition, il-6 expression was high at 8 hours after LPS stimulation in peripheral blood lymphocytes. In the immunohistochemistry, the greatest numbers of cells were recognized by grouper IL-6 antiserum in the head kidney at 8 hours after LPS stimulation. The prokaryotically produced recombinant grouper IL-6 (rIL-6) was a 28 KDa protein. At the end of this study, an E. coli expressed rIL-6 was injected into the test fish, and the immune related gene expression at different times and in various organs was evaluated and to determine the biofunction of grouper IL-6. The data showed the pre-inflammatory cytokines, tnf-α1, tnf-α2 and il-1β, as well as the Th1 specific transcription factor, t-bet, were down-regulated. The Th2 specific transcription factors, c-maf and gata, as well as the Ig were up-regulated in immune organ after rIL-6 injection. We also observed that the acute-phase protein gene, crp, was up-regulated in live. These results indicate that grouper IL-6 plays an important role in the immune system and is a regulator for naїve T helper cell differentiation towards Th2 cells, because it elicited antibody production.

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