臺灣博碩士論文加值系統

惡性的腫瘤細胞會將其生長代謝的方式由傳統的檸檬酸循環 (TCA cycle)，轉變成較快速的糖解作用 (Glycolysis) 來獲取能量；這個現象稱為瓦氏效應 (Warburg effect)。過去研究指出代謝酵素的表現異常、有氧呼吸電子傳遞鏈的缺失、缺氧、酸性的微環境，都會使腫瘤細胞能量代謝轉移到糖解作用變得更惡性。乳酸脫氫酶 (Lactate dehydrogenase) 的主要功能為丙酮酸 (Pyruvate) 與乳酸 (Lactate) 之間的轉換，這個反應會伴隨著菸鹼醯胺腺嘌呤 (NADH) 的氧化與還原，進而去調控細胞能量代謝與抑癌基因p53的表現。乳酸脫氫酶是由兩種基因乳酸脫氫酶甲型與乙型 (LDHA, LDHB) 轉譯出五種不同的異構物。乳酸脫氫酶甲型常在腫瘤細胞中被大量表現，被認為與維持腫瘤細胞生長有關係；乳酸脫氫酶乙型在近幾年研究指出在腫瘤細胞中會有默化的現象，但詳細的機制則不清楚。為了解乳酸脫氫酶乙型在腫瘤細胞中扮演的角色、以及對其能量代謝的影響，本研究在子宮頸癌HeLa細胞株中穩定抑制乳酸脫氫酶乙型的表現。由實驗結果可發現，缺失乳酸脫氫酶乙型表現的細胞株不論增生或爬行的能力都獲得了提高。在表現變得更惡性得同時，我透過西方墨點法 (Western blot) 分析發現，該細胞株檸檬酸循環與電子傳遞鏈 (OHPHOS) 的酵素表現量皆有異常。經由流式細胞儀分析也可發現氧化代謝物 (ROS) 及H2O2的產生減少且粒線體膜電位 (Membrane potential) 大幅降低。暗示著這株細胞株能量代謝偏向利用糖解作用來獲得能量；限制細胞培養基裡的葡萄糖濃度 (Glucose deprivation)，也可以發現喪失乳酸脫氫酶乙型表現的細胞株對此改變較為敏感，而大量走向細胞凋亡 (Apoptosis)。為了更進一步了解乳酸脫氫酶乙型是透過什麼作用機制導致腫瘤細胞的癌化，我利用了各種不同的化癌藥物 (2-Deoxyglucose, Etoposide)、放射線治療 (Radiation) 等方法處理該細胞株，皆發現較為惡性的抑制乳酸脫氫酶乙型的細胞株有較好的生存能力。處理白藜蘆醇 (Resveratrol) 可發現該細胞株對此藥物較為敏感，在極低濃度下細胞就會大量走向細胞凋亡。經由西方墨點法可發現，白藜蘆醇能抑制該細胞株六碳醣磷酸酶1（Hexokinase I）的作用，使細胞株無法進行糖解作用而死亡。本篇研究證實在子宮頸癌細胞株抑制乳酸脫氫酶乙型的表現，可以造成腫瘤細胞的惡化，也透過西方墨點法及藥物處理的實驗解釋了該細胞株透過瓦式效應促使腫瘤細胞轉型 (Transformation) 的機制。並且也提供了一個新穎的藥物治療標地去專一性地殺死此種類型的腫瘤細胞。

Malignant tumor obtains energy by Warburg effect, shifting its energy metabolism from traditional Tricarboxylic acid cycle (TCA cycle) to aerobic glycolysis. Dysregulation in metabolic enzymes control, defects in oxidative phosphorylation, hypoxia and acidic microenvironment are some of the possible factors that could cause Warburg effect. Lactate dehydrogenase (LDH) is a tetrameric enzyme composed by two different genes transcribes, LDHA and LDHB. LDH catalyzes the reversible conversion of pyruvate and lactate, with NADH and NAD+ oxidoreduction. Recent studies show that LDHA favors converting pyruvate to lactate and produces NAD+. Under this condition, the tumor suppressor gene p53 would be no longer stable, and the microenvironment would become acidic, which benefits tumor growth. On the opposite of that, LDHB seems to play a distinct role in tumor development. Previous research has indicated that LDHB would be downregulated in tumor cells, although the exact mechanism is still unknown. To approach more details and insightful results, I have established a stable knockdown LDHB in HeLa cell line by applying the RNAi technique. The analysis of the phenotype of this knockdown cell line showed that loss of LDHB expression would transform the tumor cells to a more malignant stage, including cell proliferation rate acceleration and cell migration ability enhancement. Besides the more malignant condition, western blot analysis indicated that loss of LDHB function results in aberrant TCA cycle and glycolysis enzymes expression and oxidative phosphorylation complexes dysregulation. Furthermore, flow cytometry assays showed that this cell had produced a low level of ROS and H2O2, and had a serious defect in mitochondria membrane potential. These results suggest that knockdown of LDHB in HeLa cell would shift its energy metabolism to glycolysis. To test this hypothesis, I cultured the cell under glucose deprivation condition, and the result showed that knockdown of LDHB cell line was sensitive to glucose concentration, confirming that the cell was under glucose dependent growth. In order to study the metabolic regulation in more depth, I used 2-deoxyglucose, etoposide, radiation and resveratrol to treat the cells. Surprisingly, malignant knockdown LDHB cells exhibited higher resistance to those treatments, which is a normal phenomenon for malignant cancer cell, but it is more sensitive to low dose resveratrol treatment. Further examination of underline mechanism, points out that resveratrol would downregulate hexokinase I expreesion, which is a key enzyme for glycolysis metabolism. This research tests that loss of LDHB expression results in malignant tumor transformation. Furthermore, phenotype change is affected by the alteration of energy metabolism, namely the Warburg effect. This research also proposed that resveratrol specific inihibits hexokinase I expression and blocks glycolysis pathway, that might be a specific target for cancer therapy.

第一章 緒論
1-1 瓦氏效應與惡性腫瘤細胞的能量代謝…………………………11
1-2 腫瘤細胞的轉型…………………………………………………13
1-3 糖解作用與電子傳遞鏈在惡性腫瘤細胞的調控………………15
1-4 乳酸脫氫酶 (Lactate dehydrogenase) ...………………………..19
1-5 白藜蘆醇 (Resveratrol)………………………………………….20
1-6 研究動機……………………………………………….………...21
第二章 實驗材料及方法
2-1 實驗材料………………………………………………..23
2-1-1 勝任細胞株……………………………………………………23
2-1-2 限制酶………………………………………………………....23
2-1-3 載體……………………………………………………………23
2-1-4 細胞株…………………………………………………………23
2-1-5 化學藥品………………………………………………………24
2-1-6 試劑……………………………………………………………27
2-1-7 抗體……………………………………………………………27
2-1-8 培養液…………………………………………………………29
2-1-9 細菌用的培養基- LBA plate………………………………….30
2-1-10 緩衝液…………………………………………………………31
2-1-11 各種試劑配製…………………………………………………38
2-1-12 勝任細胞之製備………………………………………………40
2-1-13 儀器設備………………………………………………………40
2-2 實驗方法………………………………………………..41
2-2-1 基本分子生物技術……………………………………...41
2-2-1.1 質體的製備…………………………………………………..41
2-2-1.2 構築質體的方法……………………………………………..43
2-2-2 細胞培養………………………………………………...46
2-2-2.1 細胞株的繼代………………………………………………..46
2-2-2.2 細胞數目的計數……………………………………………..47
2-2-2.3 細胞的冷凍儲存……………………………………………..48
2-2-2.4 解凍細胞…………………………...………………………...48
2-2-2.5 短暫性轉染 (Transient transfection)………………………...49
2-2-2.6 建立持續抑制 LDHB 的穩定細胞株 (stable cell lines)…..49
2-2-2.7 細胞處理化學藥物、放射線實驗…………………………...49
2-2-3 細胞表現型分析相關實驗……………………………...50
2-2-3.1 細胞型態觀察實驗…………………………………………..50
2-2-3.2 細胞生長觀察實驗…………………………………………..50
2-2-3.3 細胞增殖分析 (MTT assay)………………………………...50
2-2-3.4 溴脫氧尿核苷酸混合實驗 (BrdU incorporation assay)……51
2-2-3.5 細胞集落形成法 (Colony formation assay)………………...51
2-2-3.6 軟洋菜膠細胞集落形成法 (Soft agar colony formation assay)..............................................52
2-2-3.7 乳酸脫氫酶活性測定 (LDH activity assay)………………...52
2-2-3.8 細胞培養液pH值測定………………………………………53
2-2-3.9 細胞內ATP含量測定………………………………………..53
2-2-3.10 細胞內ROS測定…………………………………………….54
2-2-3.11 細胞內H2O2測定………………………………………….....55
2-2-3.12 粒線體膜電位測定 (Mitochondria membrane potential)…...55
2-2-3.13 傷口癒合爬行能力測定 (Wound healing migration assay)...55
2-2-3.14 細胞爬行能力測定 (Boyden chamber cell migration assay).56
2-2-3.15 明膠酶基質金屬蛋白酵素測定 (Zymography MMP assay).57
2-2-3.16 細胞週期測定 (Cell cycle)………………………………….57
2-2-3.17 Propidium Iodide (PI) – Annexin V 雙染實驗……………...58
2-2-4 實驗質體的構築方法…………………………………...59
2-2-5 西方墨點法 (Western blotting)…………………………60
2-2-6 免疫螢光染色分析 (Immunofluorescence staining)…...61
2-2-7 F-actin染色 (Phalloidin staining)………………………62
2-2-8 其他實驗………………………………………………...63
2-2-8.1 雙重冷光基因活性的測定 (Dual- Luciferase assay)…….…63
2-2-8.2 蛋白質定量 (Micro BCATM protein assay reagent kit)……...63
2-2-8.3 觀察細胞綠螢光表現量……………………………………..63
第三章 實驗結果
3-1 分析LDHA和LDHB在不同的正常組織及腫瘤細胞的表現量…………………………………………………………………64
3-2 在HeLa細胞株建立穩定默化LDHB的細胞株………………65
3-3 喪失LDHB表現的細胞株粒線體功能出現缺失……………...66
3-4 喪失LDHB表現的細胞株因瓦氏效應轉變成惡性的腫瘤細胞…………………………………………………………………67
3-5 化療藥物2-DG、Etoposide、ATP與放射線治療無法專一性 地殺死惡性的shLDHB細胞株…………………………………70
3-6 Resveratrol可以透過抑制細胞糖解作用酵素Hexokinase I來專一性地殺死shLDHB細胞株……………………………………72
第四章 討論
4-1 總結………………………………………………………………75
4-2 Resveratrol是如何抑制Hexokinase I的表現量……………….75
4-3 NADH與NAD+ 與p53的關係………………………………...77
4-4 利用LC-MS分析HeLa代謝體的變化………………………..78
4-5 瓦氏效應與脂質的合成………………………………………...79
第五章 參考文獻……………………………………82
第六章 實驗結果圖表………………………………94

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