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研究生:林裔恬
研究生(外文):LIN,YI-TIAN
論文名稱:探討於能量壓力下Grail對細胞的調控角色
論文名稱(外文):To investigate the role of Grail in the regulation of cellular energy stress response
指導教授:陳英傳
指導教授(外文):Chen, Ying-Chuan
口試委員:陳明晃林子暘洪東源蔡文銓
口試日期:2019-05-02
學位類別:碩士
校院名稱:國防醫學院
系所名稱:生理學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2019
畢業學年度:107
語文別:中文
論文頁數:83
外文關鍵詞:Grailenergy stressglucose starvation induce cell death
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葡萄糖為生物體中重要的養分來源,藉由糖解作用進入到克氏循環(TCA cycle)產生能量供給於細胞使用,藉此幫助細胞生長及存活,除此之外陸續有文獻指出葡萄糖對於癌細胞的遷徙 (migration) 及發展(progression)有密切關聯。而當葡萄糖缺乏(glucose starvation)情況下會造成細胞型態的改變及細胞死亡。癌細胞會藉由改變代謝路徑去適應環境壓力,且癌細胞比正常細胞對葡萄糖有更高的需求量,並傾向於使用糖解作用取代克氏循環,藉此快速產生能量及乳酸(lactate)幫助腫瘤生長。
AMPK為細胞內重要的能量恆定調節蛋白,當細胞處於葡萄糖缺乏條件下,會透過活化p53去啟動AMPK信號傳遞路徑左右細胞存活。於我們實驗室之前的研究中發現一個全新的p53結合蛋白 Grail,它可以去調控DNA損傷下p53所誘發的細胞死亡。除此之外,我們實驗室於最近的研究也發現Grail會調控脂肪細胞分化與高脂飲食所誘導的肥胖。由這些研究結果可以得知Grail在能量代謝與調控細胞死亡中扮演重要角色,因此於本篇論文中想進一步探討 Grail與能量缺乏所誘發的細胞死亡之間的關聯性及可能參與的分子機轉。
在本篇論文實驗中發現細胞處於能量缺乏的條件下,過度表現Grail會促進能量缺乏所誘導的細胞死亡,並增加AMPK蛋白的磷酸化、糖解作用相關基因的表現及活性氧化物質( Reactive oxygen species, ROS)的產生。相反地,降低內生性Grail表現則會減少能量缺乏所誘發的細胞死亡,並降低AMPK蛋白的磷酸化、糖解作用相關基因的表現及活性氧化物質的產生。經由目前的實驗結果知道Grail對於能量缺乏壓力下所造成的細胞損傷上扮演一個重要調控角色,未來我們也將更進一步探討可能參與的傳遞路徑及分子機轉。

Glucose services as a critical source for cells, it can be converted to ATP to support cell proliferation and survival. It has been shown that glucose uptake and glycolysis can be obviously increased in cancer cells, and glucose deprivation can induce cell death in some cancer cell types. AMP-activated protein kinase (AMPK), as a well-known cellular energy sensor, is activated by p53 signaling pathway under glucose starvation condition. In previous study, our lab had demonstrated that Grail (Gene related to anergy in lymphocytes) was a novel p53 binging protein, and the evidences also revealed that Grail not only regulated p53-mediated cell cycle arrest and apoptosis in response to DNA damage but also played a crucial role of adipogenesis and diet induced obesity.
However, the role of Grail in the regulation of cellular energy stress induced cellular response is poorly understood. Our preliminary data showed that overexpression of Grail could promote glucose starvation-induced cell death, AMPK activation, expression of glycolysis related genes and levels of ROS (reactive oxygen species). Conversely, silencing of Grail expression could inhibited glucose starvation-induced cell death, AMPK activation, expression of glycolysis related genes and production of ROS under cellular energy stress. In this study, we demonstrate that Grail have an important role in the regulation of cellular energy response, and we will further investigate Grail-dependent mechanisms involving cellular energy stress-induced cell death.

目錄 I
圖目錄 III
縮寫表 VI
中文摘要 VII
英文摘要 IX
第一章 、實驗背景 1
第一節 、葡萄糖對細胞的重要性 1
壹、 細胞與腫瘤細胞對葡萄糖的依存性 2
第二節 、能量缺乏的調節者AMP-ACTIVATED PROTEIN KINASE (AMPK) 6
第三節 、何謂GRAIL ( GENE RELATED TO ANERGY IN LYMPHOCYTES ) 7
壹、 Grail的結構及相關功能 7
貳、 Grail與疾病之間的關係 8
第二章 、實驗動機 11
第三章 、實驗目的 11
第四章 、材料方法 11
第一節 、實驗細胞 11
第二節 、實驗材料或藥物 12
第三節 、實驗儀器 13
第四節 、實驗設計 14
第五節 、實驗分析與方法 15
壹、 建立stable line 15
貳、 即時定量聚合酶鏈鎖反應 (Real-time -qPCR) 16
參、 西方墨點法(Western blot anaylsis) 20
肆、 細胞計數與細胞存活檢測(MTT cell viability assay) 22
伍、 螢光顯微鏡 23
陸、 流式細胞儀 23
第五章 、實驗結果 25
第一節 、觀察在葡萄糖缺乏的條件下GRAIL MRNA表現量的變化 25
第二節 、觀察GRAIL於葡萄糖缺乏的條件下對細胞型態、細胞生存的影響及相關分子機制的影響 26
壹、 Grail於葡萄糖缺乏的條件下會降低細胞的存活率 26
貳、 Grail於葡萄糖缺乏的條件下會增加細胞的死亡率 29
參、 Grail於葡萄糖缺乏的條件下會增加凋亡蛋白Cleaved caspase 3的表現 31
肆、 Grail於葡萄糖缺乏的條件下會增加AMPK磷酸化 32
第三節 、觀察GRAIL於葡萄糖缺乏的條件下對於葡萄糖代謝的相關影響 36
第六章 、討論 38
第七章 、結論 41
第八章 、參考資料 42
第九章 、實驗結果附圖 45


Carol C. Cunningham, C. G. V. H. (2003). Energy Availability and Alcohol–Related Liver Pathology. National Institute on Alcohol Abuse and Alcoholism grants.
Chen, Y. C., Chan, J. Y., Chiu, Y. L., Liu, S. T., Lozano, G., Wang, S. L., . . . Huang, S. M. (2013). Grail as a molecular determinant for the functions of the tumor suppressor p53 in tumorigenesis. Cell Death Differ, 20(5), 732-743. Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/23370271.
Cho, E. S., Cha, Y. H., Kim, H. S., Kim, N. H., & Yook, J. I. (2018). The Pentose Phosphate Pathway as a Potential Target for Cancer Therapy. Biomolecules & Therapeutics, 26(1), 29-38.
Crowley, L. C., & Waterhouse, N. J. (2016). Detecting Cleaved Caspase-3 in Apoptotic Cells by Flow Cytometry. Cold Spring Harb Protoc, 2016(11).
El Mjiyad, N., Caro-Maldonado, A., Ramirez-Peinado, S., & Munoz-Pinedo, C. (2011). Sugar-free approaches to cancer cell killing. Oncogene, 30(3), 253-264.
Fadaka, A., Ajiboye, B., Ojo, O., Adewale, O., Olayide, I., & Emuowhochere, R. (2017). Biology of glucose metabolization in cancer cells. Journal of Oncological Sciences, 3(2), 45-51.
Ferrier, D. R. (2015). Lippincott's lllustrated Reviews: Biochemistry. SIXTH EDITION.
Ha, J., Guan, K. L., & Kim, J. (2015). AMPK and autophagy in glucose/glycogen metabolism. Mol Aspects Med, 46, 46-62. Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/26297963.
Haymaker, C., Yang, Y., Wang, J., Zou, Q., Sahoo, A., Alekseev, A., Nurieva, R. (2017). Absence of Grail promotes CD8(+) T cell anti-tumour activity. Nat Commun, 8(1), 239. Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/28798332.
Hirayama, A., Kami, K., Sugimoto, M., Sugawara, M., Toki, N., Onozuka, H., Soga, T. (2009). Quantitative metabolome profiling of colon and stomach cancer microenvironment by capillary electrophoresis time-of-flight mass spectrometry. Cancer Res, 69(11), 4918-4925. Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/19458066.
Hoeller, D., Hecker, C.-M., & Dikic, I. (2006). Ubiquitin and ubiquitin-like proteins in cancer pathogenesis. Nature Reviews Cancer, 6, 776. Retrieved from https://doi.org/10.1038/nrc1994.
Iurlaro, R., Püschel, F., León-Annicchiarico, C. L., O'Connor, H., Martin, S. J., Palou-Gramón, D., Muñoz-Pinedo, C. (2017). Glucose Deprivation Induces ATF4-Mediated Apoptosis through TRAIL Death Receptors. Molecular and Cellular Biology, 37(10), e00479-00416. Retrieved from https://mcb.asm.org/content/mcb/37/10/e00479-16.full.pdf.
Kuehne, A., Emmert, H., Soehle, J., Winnefeld, M., Fischer, F., Wenck, H., Zamboni, N. (2015). Acute Activation of Oxidative Pentose Phosphate Pathway as First-Line Response to Oxidative Stress in Human Skin Cells. Mol Cell, 59(3), 359-371. Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/26190262.
Liberti, M. V., & Locasale, J. W. (2016). The Warburg Effect: How Does it Benefit Cancer Cells? Trends Biochem Sci, 41(3), 211-218. Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/26778478.
Lin, H. T., Chen, C. C., Liu, P. Y., Wu, H. L., Wu, T. H., Huang, C. H., & Chen, Y. C. (2018). Grail attenuates influenza A virus infection and pathogenesis by inhibiting viral nucleoprotein. Sci Rep, 8(1), 17242. Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/30467324.
Liu, P., Hsieh, P., Lin, H., Liu, T., Wu, H., Chen, C., & Chen, Y. (2018). Grail is involved in adipocyte differentiation and diet-induced obesity. Cell Death Dis, 9(5), 525. Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/29743578.
Lu, J., Tan, M., & Cai, Q. (2015). The Warburg effect in tumor progression: mitochondrial oxidative metabolism as an anti-metastasis mechanism. Cancer Lett, 356(2 Pt A), 156-164. Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/24732809.
Nakamichi, S., Senga, Y., Inoue, H., Emi, A., Matsuki, Y., Watanabe, E., Kasuga, M. (2009). Role of the E3 ubiquitin ligase gene related to anergy in lymphocytes in glucose and lipid metabolism in the liver. J Mol Endocrinol, 42(2), 161-169. Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/19060180.
Niroshana Anandasabapathy, G. S. F., Debra Bloom, C. H., Violette Paragas, C. S., Heidi Skrenta, M. H., & C. Garrison Fathman, a. L. S. (2003). GRAIL: An E3 Ubiquitin Ligase that Inhibits Cytokine Gene Transcription Is Expressed in Anergic CD4 T Cells. Immunity.
Patra, K. C., & Hay, N. (2014). The pentose phosphate pathway and cancer. Trends Biochem Sci, 39(8), 347-354. Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/25037503.
Salvatore Papa , P. M. C., Concetta Bubici (2019). . Oncogene.
Stincone, A., Prigione, A., Cramer, T., Wamelink, M. M., Campbell, K., Cheung, E., . . . Ralser, M. (2015). The return of metabolism: biochemistry and physiology of the pentose phosphate pathway. Biol Rev Camb Philos Soc, 90(3), 927-963. Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/25243985.
Vincent, E. E., Coelho, P. P., Blagih, J., Griss, T., Viollet, B., & Jones, R. G. (2015). Differential effects of AMPK agonists on cell growth and metabolism. Oncogene, 34(28), 3627-3639. Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/25241895.
Whiting, C. C., Su, L. L., Lin, J. T., & Fathman, C. G. (2011). GRAIL: a unique mediator of CD4 T-lymphocyte unresponsiveness. FEBS J, 278(1), 47-58. Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/21078124.
Yun, H., Kim, H. S., Lee, S., Kang, I., Kim, S. S., Choe, W., & Ha, J. (2009). AMP kinase signaling determines whether c-Jun N-terminal kinase promotes survival or apoptosis during glucose deprivation. Carcinogenesis, 30(3), 529-537. Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/19037093.
Zhang, C. S., Hawley, S. A., Zong, Y., Li, M., Wang, Z., Gray, A., . . . Lin, S. C. (2017). Fructose-1,6-bisphosphate and aldolase mediate glucose sensing by AMPK. Nature, 548(7665), 112-116. Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/28723898.
Zhang, M., Liu, T., Sun, H., Weng, W., Zhang, Q., Liu, C., . . . Sheng, W. (2018). Pim1 supports human colorectal cancer growth during glucose deprivation by enhancing the Warburg effect. Cancer Sci, 109(5), 1468-1479.
Zhao, Y., Hu, X., Liu, Y., Dong, S., Wen, Z., He, W., . . . Shi, M. (2017). ROS signaling under metabolic stress: cross-talk between AMPK and AKT pathway. Mol Cancer, 16(1), 79. Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/28407774.
Zhao, Y., Wieman, H. L., Jacobs, S. R., & Rathmell, J. C. (2008). Mechanisms and methods in glucose metabolism and cell death. Methods Enzymol, 442, 439-457.














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