黏液纖維肉瘤是常見具黏液型態的軟組織肉瘤。臨床上，黏液纖維肉瘤復發時常會增加腫瘤惡化等級，最終導致轉移。而現今轉移性黏液纖維肉瘤難以治療，是構成肉瘤相關死亡的主要原因。根據本研究團隊先前應用免疫組織化學染色法分析黏液軟組織肉瘤檢體結果，發現p21 protein (Cdc42/Rac)-activated kinase 1（PAK1）蛋白質有高度表現的情形。PAK1為絲胺酸/蘇胺酸激酶（serine/threonine kinase），可受不同的訊息傳遞路徑刺激，調控細胞存活、進行有絲分裂與細胞骨架重組等。首先，我們測試不同的黏液肉瘤細胞株，包含OH931、NMFH1和NMFH2之PAK1基因內生性mRNA和蛋白質表現，發現PAK1高度表現於OH931和NMFH1；而低表現於NMFH2細胞株。我們更進一步利用wound healing與matrigel transwell assay發現，將帶有wild-type PAK1基因或PAK1 T423E突變株的質體，轉殖入NMFH2細胞可促進細胞遷移和侵犯能力。相反的，以short hairpin RNA干擾技術抑制PAK1基因表現，則會抑制NMFH1細胞遷移速率和侵犯能力。此外，5-bromo-2-deoxyuridine assay和colony formation assay發現，不管送入外源性PAK1表現質體使其高度表現或抑制PAK1基因皆會影響細胞的生長與轉型情形。令人意外地，以hepatocyte growth factor處理NMFH2細胞，誘導PAK1 (Thr212) 磷酸化並使其進入細胞核。總結以上，PAK1基因在黏液肉瘤中扮演著致癌的角色。

The common type of myxoid soft tissue sarcomas is myxofibrosarcoma. Clinically, increased tumor grading and staging are frequently observed in myxofibrosarcomas after relentless local recurrences, which may eventually lead to metastatic diseases. However, metastatic myxofibrosarcomas are often refractory to current treatment strategies and constitute the primary cause of sarcoma-related death. Immunohistochemistry staining was applied to analyze myxoid tumors of soft tissue in our previous studies, and p21 protein (Cdc42/Rac)-activated kinase 1 (PAK1) was identified to be significantly upregulated in myxoid soft tissue sarcomas. The PAK1 is a pivotal serine/threonine kinase, which integrates stimuli from various signaling pathways to regulate cell survival, mitosis and cytoskeletal remodeling, etc. We first examined the endogenous PAK1 mRNA and total PAK1 protein levels in various myxoid sarcoma cell lines, including OH931, NMFH1 and NMFH2. This initial screening detected that upregulated PAK1 expression in OH931 and NMFH1, whereas downregulated PAK1 in NMFH2 cells. By wound healing and matrigel transwell assay, we further found that transfection of the expression plasmid carrying wild-type PAK1 gene or PAK1 T423E mutant promoted cell migration and invasion abilities in NMFH2 cells. On the other hand, knockdown of the PAK1 gene by short hairpin RNA interference inhibited the migration rate and invasion ability in NMFH1 cells. By 5-bromo-2-deoxyuridine assay and colony formation assay, we found that either exogenous expression of PAK1 protein or knockdown of PAK1 gene affected cell proliferation and transformation. Interestingly, immunofluorescence demonstrated that treatment with hepatocyte growth factor induced phosphorylation of PAK1 (Thr212) and promoted its nuclear import in NMFH2 cells. In summary, PAK1 plays oncogenic roles in myxoid sarcoma carcinogenesis.

論文審定書 i
誌謝ii
摘要iii
Abstract iv
英文縮寫表v
壹、緒論（Introduction） 1
貳、實驗材料及方法（Materials and methods） 6
参、結果（Results） 20
肆、討論（Discussion） 34
伍、參考文獻（References） 36
附錄42

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