中文摘要
　　STAT為一群轉錄因子，它們可以經由許多不同的細胞激素和生長因子刺激而活化。其中，持續活化態的STAT3在人類癌症常常可見，被認為可以促進腫瘤細胞的存活以及幫助逃避免疫系統的攻擊。另一方面，STAT家族的另一成員STAT1則有著與STAT3不同的功能；STAT1被認為可以促進細胞凋亡並且活化一些發炎相關的免疫反應。被活化之後的STAT1及STAT3可以分別形成同體異構物(STAT1: STAT1或是STAT3: STAT3)或是異體異構物(STAT1: STAT3)。STAT1: STAT1同體異構物和STAT3: STAT3同體異構物在調節基因時，常常結合在相似但是不相同的元素，這點也許可以解釋為何STAT1與STAT3具有孑然不同的生理功能。然而，STAT1與STAT3如何調節腫瘤細胞內免疫反應仍不清楚。在本研究中，我們假設在腫瘤細胞中，STAT1與STAT3之間的抗衡將會影響免疫反應的走向。我們以老鼠肺癌細胞Lewis lung carcinoma (LL2)做為腫瘤細胞使用。由於LL2細胞有表現持續活化態的STAT3，因此我們利用Lipofectamine轉染技術將STAT1送入LL2細胞內。我們建立了兩種細胞株: LL2-S1C及LL2-S1；前者過度表現持續活化態的STAT1 (STAT1C)，後者過度表現單體態的STAT1蛋白。給予Interferon-(IFN-) 刺激發現，LL2、LL2-S1C及LL2-S1的細胞生長都不會被抑制，相反地，IFN-讓它們的生長更好。另一方面，給予Interleukin-6 (IL-6) 對於這些細胞的生長則沒有影響。進一步我們將這些細胞株分別經由皮下注射打入動物體內，觀察它們形成的腫瘤，發現LL2-S1C細胞明顯長的比較好。 接下來，我們利用西方點墨法檢測這些細胞株在經由IFN-或是IL-6刺激之後，細胞內STAT蛋白的表現；我們發現LL2-S1C細胞在IFN-刺激後，STAT5異常的活化；在IL-6刺激之後，LL2-S1C細胞有STAT1異常活化。為了進一步釐清由IFN-刺激之後造成的細胞增生，我們檢測了STAT5所調控的兩個下游基因: bcl-2及cyclin D1。而我們發現這二個蛋白的表現並沒有因為IFN-刺激而受到影響。另一方面，我們推論STAT1C在LL2-S1C細胞內可能與一些特定的蛋白結合而影響了STAT家族的活化，以致於在IFN-或是IL-6刺激之後會有異常的STAT蛋白被活化；因此，我們收集LL2、LL2-S1C及LL2-S1的細胞蛋白，經由免疫沉澱將與STAT1結合的蛋白分離之後，利用二維電泳 (2D-PAGE)以及蛋白質譜分析—MALDI-MS (Matrix-assisted Laser Desorption/Ionization-Mass Spectrometry)區分出特定表現在LL2-S1C的沉澱蛋白內的因子—alkaline phosphatase 3。而在這個系統中，alkaline phosphatase 3的生物功能需要進一步研究才能釐清。經由蘇木紫伊紅染色，我們發現在LL2-S1C及LL2-S1所產生的腫瘤中，浸潤的嗜中性球明顯比LL2所產生的腫瘤多。根據此結果，我們推論在表現持續活態STAT3的腫瘤細胞中，大量表現STAT1蛋白(不論是活化或非活化態)可能可以讓宿主的先天性免疫反應被回復。

　　在本研究中，我們發現，在表現持續活化態STAT3的腫瘤細胞中，給予持續活化態STAT1的過度表現，可能回復宿主的先天性免疫系統，然而在動物體內腫瘤的生長不會被抑制。我們偶然發現送入細胞內的STAT1C過量表現會造成LL2 細胞異常的STAT蛋白活化，進一步研究其機制也許能發現一些新的JAK/STAT訊息傳遞路徑的調節系統。

Abstract
　STATs are a family of transcription factors that activated by various cytokine and growth factors. Among them, STAT3 is frequently activated in human tumors and implicated in promoting tumor cell survival and immune evasion. On the contrary, STAT1 is known to promote tumor cell apoptosis and activate immune responses. Activated STAT1 and STAT3 could form homodimer by themselves or heterodimer between each other. STAT1 and STAT3 homodimers bind selectively to very similar but not identical elements of genes, which is likely to account for their very different biological effects. However, how STAT1 and STAT3 cooperatively regulate tumor immune responses is still not clear. We hypothesize that the balance between STAT1 and STAT3 may influence tumor immune responses in cancer cells. In this study, we chose mouse Lewis lung carcinoma (LL2) cells as the tumor model. STAT3 is constitutively activated in LL2 cells. We have transfected LL2 cells with STAT1C (an active form of STAT1) and selected clones (LL2-S1C) those stably expressed the protein for further studies. Also we generated LL2-S1 cells which stably expressed free form STAT1 protein. Interferon- (IFN-) did not inhibit growth of LL2, LL2-S1 and LL2-S1C cells, and on the contrary, IFN-enhances growth of them. Interleulin-6 (IL-6) did not influence growth of these cells. LL2-S1C grew slower in vitro but faster in vivo than parental cells. Further we found that IFN- aberrantly activated STAT5 in LL2-S1C cells, but not LL2-S1 cells. And IL-6 aberrantly activated STAT1 in LL2-STAT1C cells. To elucidate why IFN--induced cell proliferation, we checked the expressions of bcl-2 and cyclin D1 that are downstream factors of STAT5. IFN- did not alter the expressions of both proteins. On the other hand, we infer that sequestration of some negative regulators of STAT by STAT1C may be the mechanism by which the specificity of STAT protein activation by receptor was altered. Celluar proteins of LL2, LL2-S1 and LL2-S1C cells were isolated and subjected to immunoprecipitation. The immunoprecipitated proteins were then separated by two-dimension gel electrophresis. Differentially expressed protein spots were picked up for mass spectrometry analysis. We identify that alkaline phosphatase 3 is differentially expressed in LL2-S1C proteins. The biologic function of alkaline phosphatase 3 in this system requires further studies.

　There are only few neutrophils found in LL2 subcutaneous tumor, whereas, infiltration of neutrophils was easily found in LL2-S1 and LL2-S1C tumors. The results suggest increase STAT1 (free or active form) expression in a STAT3 active cancer cells may restore the innate immunity of host.

　In this study, we found that over expression of STAT1 in LL2 cells that express constitutively active STAT3 may restore innate immunity but did not inhibit the tumor formation, especially in vivo. We incidentally found that STAT1C overexpression cause aberrant STAT activation in LL2 cells. Further studies on the underlying mechanisms may explore a novel regulatory system of JAK/STAT pathway.

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