前言:腸胃道基質瘤(Gastrointestinal stromal tumors, 簡稱GIST)為腸胃道最常見肉瘤(sarcoma)之一。這種肉瘤起源於腸胃道平滑肌層的神經叢細胞── Interstitial cells of Cajal的不正常增生，而且80%以上的GIST具有c-KIT gene的變異，導致所轉碼的receptor tyrosine kinase出現異常的活化。
腫瘤細胞由小型增長成晚期大型腫瘤的過程中，會面臨低氧(hypoxia)的微環境變化，原因是大型腫瘤內部的細胞較無法接收到外圍血管所供應的氧氣所致。低氧微環境會對腫瘤細胞的生長、凋亡、移動和抗藥性等產生不同程度的影響。目前低氧微環境對GIST影響的研究較少，本研究主要探討在低氧環境對GIST腫瘤細胞株的生長與死亡影響，與相關的因應機制。
材料及方法: 分別用三種GIST細胞株(882、62、48b) 和GIST病人的檢體作為實驗材料。首先，將手術切除的GIST腫瘤組織分為中心部分(central part)和周圍的部分(peripheral part)，以西方墨點試驗(western blotanalysis) 和組織免疫染色(IHC)實驗，分析這兩部分所表現生物訊號的差異。再者，將GIST細胞株放在低氧培養箱一至三天，用不同assays觀察其細胞存活率、凋亡率，並以western blot分析其分子訊號表現。
結果: 首先，吾人分析手術切除的腫瘤，發現較大的GIST(7.5cm~15cm)中心部分的HIF-1α以及p-Kit表現比周圍部分多。再以MTT assay分析細胞存活。在低氧箱培養1-3天後， GIST 882比另2種GIST細胞株(62、48b)具有較高的存活率。而且， 西方墨點試驗顯示在低氧培養下的GIST882的p-KIT, p-AKT及p-PKCθ有較明顯的活化。目前已知p-AKT及p-PKCθ分別會對BAD的serine 136、112位置進行磷酸化，使之活性降低，進而減少細胞凋亡。因此使用MTT, western blot, Flow cytometry和TUNEL assay等方法，觀察在低氧環境下，對GIST882細胞株施以AKT的抑制劑(MK2206)或PKCθ的抑制劑(Sotrastaurin)的影響。結果顯示MK2206可以增加細胞凋亡和減少其存活率。
結論: 較大的GIST中心部分可能出現低氧的微環境變化。GIST882細胞株在低氧環境中的生存能力較佳，可能和p-AKT的活化有關。
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Introduction: Gastrointestinal stromal tumors (GIST) are the most common sarcomas of the gastrointestinal(GI) tract. This tumor was reported to spring from the interstitla cells of Cajal, a pace-maker cells of the GI tract. About 80% GISTs harbor c-KIT gene mutation, leading to over-expression and activation on its post-translation product, a receptor tyrosine kinase.
When a tumor grows, a relative low-oxygen (hypoxia) microenvironment develop in the core of the tumor due to insufficient oxygen supply. The hypoxic microenvironment has different impact on growth, apoptosis, migration and drug resistance of the tumors. However, there is limited research about the influence of hypoxia on GISTs. In this study, we investigate the impact of hypoxia on the growth, cell death and molecular signaling of GISTs.
Methods: 3 GIST cell lines (882, 62 and 48b) and GIST tissues were used. The signaling in central and peripheral parts of excised GIST tumors were analyzed by western blot and IHC. The GIST cell lines were incubated in hypoxia incubator for 24~72h, and their viability, apoptosis, proliferation and molecular signals were assessed.
Results: The western blot and IHC results showed that large GISTs (7.5~15cm) expressed more HIF-1α and p-Kit. The MTT assay showed GIST 882 was more resistant to hypoxia than the other 2 cell lines. In addition, western blot also showed p-KIT, p-AKT and p-PKCθ were activated in GIST882. It was reported that p-AKT and p-PKCθ would lead to phosphorylation on serine136, 112 on BAD, reducing the activity of BAD and decreasing apoptosis. So, we treated GIST882 with p-AKT inhibitor (MK2206) and p-PKCθ inhibitor (Sotrastaurin) under hypoxia, and analyze the cell viability, cell death and proliferation by MTT assay, flow cytometry and TUNEL assay. The results showed MK2206 increased the apoptosis and reduced the viability of GIST 882 cells.
Conclusion: The hypoxic microenvironment developed in the central part of large GISTs. The resistance of GIST 882 to hypoxia might be related to the activation of p-AKT.

致謝 i
中文摘要 ii
英文摘要 iv
壹、緒論 1
一、腸胃道基質瘤(Gastrointestinal Stromal Tumors, GISTs) 1
二、GISTs的致癌訊息傳遞路徑(Oncogenic signaling ) 2
三、低氧環境對腫瘤之影響(The hypoxic effect on tumor) 3
四、低氧誘導分子(Hypoxia-inducible factor) 3
五、研究目的 4
貳、實驗材料 5
參、實驗方法 11
肆、實驗結果 17
伍、討論 21
陸、圖表 26
柒、參考文獻 44

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