臺灣博碩士論文加值系統

肺癌在全世界的流行病學中罹患率及致死率皆位居前兩名，高致死率可能與癌症幹細胞 (Cancer stem cells, CSCs) 相關。這群細胞具有較強的抗藥性和自我更新的能力、移動、穿透和分化能力，導致癌症容易遠端轉移，易造成癌症復發，因此癌症幹細胞是研究上很重要的一個議題。在正常細胞醣類的代謝主要經由代謝效率較佳的有氧代謝，相反的癌細胞卻傾向大量、快速的無氧代謝，這種癌細胞代謝方式的特性稱為瓦氏效應 (Warburg effect)，癌症幹細胞則更傾向無氧代謝的情況。乳酸脫氫酶 (Lactate dehydrogenase A, LDHA)在醣解進到無氧代謝的路徑中扮演關鍵角色。先前研究中發現頭頸癌及肝癌中乳酸脫氫酶的高表現和癌細胞生長有正相關，並且初步看到抑制LDHA無氧代謝能使得癌症幹細胞的形成減少。但目前為止尚未在肺癌的模式中探討代謝和癌症幹細胞的關聯，因此本研究目的是探討乳酸脫氫酶在癌症的進程以及癌症幹細胞中所扮演的角色。
首先透過免疫組織化學染色分析肺癌病人組織微陣列切片，發現在進程較後期的病人中乳酸脫氫酶的表現有上升的趨勢，並且和癌症幹細胞的標誌 (Oct4)表現量具正相關性，另外透過Suverexpress生物資訊資料庫分析，發現在兩個不同的資料庫中，乳酸脫氫酶及Oct4高表現的肺癌病人有較差的預後，以上結果顯示，乳酸脫氫酶的表現和疾病的進程成相關。接著利用兩株惡行度不同的肺腺癌細胞透過慢病毒短髮夾核糖核酸系統(Lentivirus shRNA system)來抑制乳酸脫氫酶表現(shLDHA)，結果發現癌細胞的移動及生長能力會受到抑制。進一步將癌細胞養成腫瘤微球體 (Sphere)，分析其癌症幹細胞相關特性是否受到影響，結果發現在抑制乳酸脫氫酶後其癌症幹細胞相關標誌 (Oct4, Nanog) 表現受到抑制，而腫瘤微球體形成能力也降低，並且細胞侵襲和化學治療藥物抗藥性的能力亦下降。
綜合以上結果顯示，乳酸脫氫酶可促進肺癌的發展及癌症幹細胞的生成和功能，因此在癌症的治療上可能可以透過抑制其代謝來達到抑制癌症進展的功效。

Lung cancer is the most common cause of cancer deaths globally, and several new cases are estimated to occur worldwide. The survival rate of patients with lung cancer is poor because of a small population of cells called cancer stem cells (CSCs). Certain characteristics of CSCs, including chemodrug resistance and self-renewing capacity, cause relapse. Moreover, the higher migration, invasion, and differentiation abilities of CSCs cause tumor metastasis. The Warburg effect is a phenomenon in which malignant cancer cells exhibit abnormal metabolic phenotypes. Normally, cells derive energy from aerobic metabolism because of its efficient ATP production, which requires oxygen. In contrast to malignant cells, cancer cells derive energy from enhanced aerobic glycolysis. The enzyme lactate dehydrogenase A (LDHA) catalyzes the interconversion of pyruvate and lactate. Thus, it is a key enzyme in glycolysis and anaerobic metabolism. A previous study showed that higher LDHA expression is correlated with poorer survival, higher tumor growth, and induction of CSC properties. However, the role of LDHA in nonsmall cell lung cancer (NSCLC) has not been reported, and the association between LDHA and CSC properties remains unclear. Thus, this study determined the role of LDHA in NSCLC progression.
First, through immunohistochemical staining, we observed higher LDHA expression in patients with late-stage NSCLC. We also found that LDHA expression was significantly increased in patients with lymph node metastasis. Furthermore, patients with higher Oct4 expression had higher LDHA expression, indicating the correlation between Oct4, a CSC marker, and LDHA expression. Moreover, based on the analysis of two databases, higher LDHA expression was associated with poorer survival and higher Oct4 expression. The aforementioned results indicated that high LDHA and Oct4 expression is associated with poor survival and late staging. Second, we investigated the role of LDHA in the NSCLC cell lines H1299 and A549 by using the lentivirus shRNA system to inhibit LDHA expression. The result revealed that LDHA inhibition reduced the proliferation and migration of H1299 and A549 cells. Furthermore, analyses of H1299-driven tumor spheroids showed that the downregulation of LDHA decreased the expression of CSC-associated markers, such as Oct4, Sox2, and Nanog, even at RNA and protein levels. Thus, LDHA inhibition reduced the sphere formation, migration, and invasion of CSCs.
Altogether, these findings indicated that LDHA can promote NSCLC progression and CSC properties. Thus, targeting the metabolism of CSCs may provide a novel therapeutic strategy for lung cancer. However, the metabolic mechanisms underlying the modulation of CSCs and their microenvironment, including chemokines and receptors, remain unknown.

目錄
誌謝 i
摘要 iii
Abstract v
目錄 vii
導論 Introduction 1
實驗目的Aim 14
材料與方法 Materials and methods 15
實驗方法 Experiment Methods 28
實驗結果 Results 44
討論 Discussion 53
參考文獻 Reference 56
實驗圖表 60
附錄 75

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