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研究生:張朝欽
研究生(外文):Chou-ChinChang
論文名稱:六碳醣激酶II缺陷對癌症細胞的影響
論文名稱(外文):The Effect of Hexokinase II Deficiency in Cancer Cells
指導教授:張文粲
指導教授(外文):Wen-Tsan Chang
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生物化學暨分子生物學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2016
畢業學年度:104
語文別:中文
論文頁數:95
中文關鍵詞:六碳醣激酶二2-去氧葡萄糖每福敏
外文關鍵詞:Hexokinase 22-deoxyglucoseMetformin
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在腫瘤生長的過程中會偏好利用糖解作用獲得能量並產生大量的乳酸使腫瘤周遭環境酸化,德國科學家Otto Warburg首次在1920年發現了這個現象,後來被科學家們稱為瓦氏效應。而糖解作用的第一步驟就是由六碳糖激酶
(Hexokinase, HK)將葡萄糖轉化成磷酸葡萄糖,進行下一步代謝,最終轉變成乳酸取得能量。在人類細胞中六碳糖激酶有四種不同的異構物,分別是:HK1、HK2、HK3及HK4,在大多數的正常細胞中HK1會常態性的表達扮演著啟動糖解作用的角色,HK3及HK4則是有組織特異性的表達較少被人研究,然而在過去的研究中發現許多的癌症中都有HK2的高表達,並伴隨著較高的轉移率、較差的預後及存活率。因此我利用CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeat/Cas 9 system, CRISPR/Cas 9 system) 系統剔除HK2並嘗試去釐清其在癌細胞中所扮演的角色。首先成功地在HeLa、MDA-MB-231及H1299細胞中篩選出HK2剔除的細胞株,接著對於這些細胞株作生長的分析,發現HK2剔除的細胞呈現生長變慢的現象。接著分別以觀察培養液的酸鹼值及對葡萄糖的依賴度分析其瓦氏效應的程度,實驗發現HK2剔除株對於在培養液的酸化程度及葡萄糖的依賴度有降低的現象,再來以西方墨點法分析其分子機制,發現在HK2的缺陷會降低ERK蛋白的磷酸化程度,推測可能是導致細胞生長變慢的主因。最後為了治癒癌症,選用抗癌藥物2-去氧葡萄糖 (2-Deoxyglucose, 2-DG) 及每福敏 (Metformin) 作處理,發現HK2缺陷的細胞會降低對2-DG的藥物敏感度,而相反的每福敏卻可以增強HK2缺陷細胞的藥物敏感度。綜合以上的結果HK2的缺陷會減緩癌細胞的生長及瓦氏效應的程度,且HK2的缺失會使2-DG對癌細胞作用下降,但卻會增加癌細胞對每福敏的藥物敏感度。

Normal differentiated cells mainly rely on mitochondrial oxidative phosphorylation to generate the energy required for cell progression. However, instead of mitochondria oxidative phosphorylation, most cancer cells generate energy by aerobic glycolysis, a phenomenon defined as “the Warburg effect” which was discovered by Otto Warburg in 19th century. Hexokinase (HK) is the initial enzyme in glycolysis, it primarily converts glucose into glucose-6-phosphate for the next step of glycolysis. In mammalian, there are four isoforms of HK: HK1, HK2, HK3 and HK4. HK1 is constitutively expressed in normal tissues; HK3 and HK4 are only expressed in specific tissues such as muscle and liver. In the past studies, HK2 is highly expressed in cancer cells and high level of HK2 is usually linked to high metastasis and poor prognosis. Therefore, we assumed HK2 may play a role in cancer progression. In order to clarify the role of HK2 in cancer cells, Clustered regularly-interspaced short palindromic repeats/Cas9 (CRISPR/Cas9) was used to knockout HK2 expression in HeLa (cervical cancer), MDA-MB-231 (breast cancer) and H1299 (lung cancer) cancer cell lines. First, I successfully isolated HK2 knockout cell from HeLa cells, then confirmed that HK2 deletion decreases HeLa cell growth. HK2 deficient HeLa cells showed lower glucose reliance than wild type cells by different doses of glucose treatment. To cure cancer, we used two kinds of anticancer drugs to treat HK2 deficient cells. After (2-DG) treatment, HK2 deficient cells lost drug sensitivity when compared with control HeLa cells, but opposite results are showed in Metformin treatment. These results were also confirmed in MDA-MB-231 and H1299 cell lines. Taken together, cancer cells with HK2 deficiency can decrease cell growth, glucose reliance and the sensitivity of 2DG but increased the effect on Metformin treatment.
第一章 序論................................................1
1-1 癌症...................................................1
1-2 細胞能量代謝.............................................1
1-3 瓦氏效應(Warburg effect)................................2
1-4 癌細胞葡萄糖代謝的調控機制.................................2
1-5 糖解作用代謝途徑的第一個酵素─六碳醣激酶 (Hexokinase)..........5
1-6 基因編輯工具─常間回文重複序列叢集/常間回文重複序列叢集關聯蛋白系統 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-Associated Proteins, CRISPR/Cas)............7
1-7 2-去氧葡萄糖 (2-Deoxy-D-glucose, 2-DG)..................9
1-8 每福敏 (Metformin).....................................10
1-9研究目的................................................11
第二章 實驗材料與方法.......................................12
2-1 實驗材料...............................................12
2-1-1 勝任細胞株 (Competent cell)..........................12
2-1-2 限制酶 (Restriction enzyme)..........................12
2-1-3 細胞株...............................................12
2-1-4 化學藥品.............................................13
2-1-5 試劑................................................15
2-1-6 抗體................................................16
2-1-7 培養液...............................................17
2-1-8 緩衝液...............................................18
2-1-9 各種試劑配置..........................................24
2-1-10 勝任細胞 (Competent cell) 之製備.....................25
2-1-11 儀器設備............................................26
2-2 實驗方法...............................................27
2-2-1 基本分子生物學技術.....................................27
2-2-2 細胞培養程序..........................................32
2-2-3 細胞相關實驗..........................................34
2-2-4 實驗質體的構築方法.....................................42
第三章 實驗結果............................................43
3-1 分析HK2在正常組織及癌細胞組織中的表達量.....................43
3-2 建立HK2剔除的細胞株.....................................43
3-3 觀察HK2缺失對於癌細胞生長及群落形成能力的影響................44
3-4 分析HK2缺失在瓦氏效應中的影響.............................45
3-5 分析HK2剔除株生長及代謝相關蛋白表現量.......................47
3-6 分析HK2的缺失株對2-DG藥物敏感度...........................48
3-7 缺失HK2會增加對Metformin藥物敏感度........................50
第四章 討論...............................................52
4-1 總結..................................................52
4-2 探討HK2及HK1在細胞中的關係...............................52
4-3 在HK2缺乏的細胞ERK所可能扮演的角色.........................53
第五章 參考文獻............................................54
第六章 實驗結果圖表.........................................59

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