電化學刺激對於卵巢癌細胞型態、基因與衰亡原因之研究

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研究生：	張繻文 Ju-Wen Chang
論文名稱：	電化學刺激對於卵巢癌細胞型態、基因與衰亡原因之研究 The effect of electrochemical stimulation on the morphology, genes and cell death mechanism of ovarian cancer cells
指導教授：	王復民 Fu-Ming Wang
口試委員:	王鵬惠 Peng-Hui Wang 白孟宜 Meng-Yi Bai
學位類別：	碩士 Master
系所名稱：	應用科技學院 - 應用科技研究所 Graduate Institute of Applied Science and Technology
論文出版年：	2021
畢業學年度：	109
語文別：	中文
論文頁數：	96
中文關鍵詞：	卵巢癌、癌細胞、電刺激、氧化銦錫導電玻璃片
外文關鍵詞：	ovarian cancer, cancer cell, electric stimulation, indium tin oxide
相關次數：	點閱：219 下載：1
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據世界衛生組織統計，於2020年約有二十萬人因卵巢癌而死亡，致死率以亞洲人最高，縱然治療方法日漸發展，但卵巢癌無法於早期被診斷且復發後病患對於抗癌藥物具有抗藥性等原因，進而影響治療效果與病患的生存率。近年除了藥物治療外，電脈衝化療法對於癌症治療亦是熱門的研究議題，該方法主要是透過施以適當強度之電場以研究細胞型態變化、基因表達和細胞活力等功能改變情形，分辨細胞可適應環境能力和受外界影響的程度。本研究欲以電化學反應達到電刺激之通電效果，且於實驗過程中紀錄細胞型態變化以及透過電壓數值，表現正負電流對於癌細胞之影響。我們以具高導電性且透明的氧化銦錫導電玻璃作為培養細胞之基材，並將其設置為三電極系統中之工作電極。對於細胞株施以正負0.1毫安培的電流過程，我們發現有無抗藥性之細胞株於正負電流之導電性不同，導致電流對於有無抗藥性之細胞株具不同之電化學趨勢性。我們發現正電流對於抗藥株將導致細胞衰亡，使抗藥株經正電流電刺激後之細胞存活率略高於負電流之存活率結果，顯示正電流對於抗藥株的電刺激效果較為顯著且使其存活率有效下降至四成，相較之下，抗藥株經負電流電刺激後細胞衰亡較不明顯仍其存活率達七成；我們亦以光學顯微鏡於電刺激之實驗過程中進行型態觀察，經觀察細胞於正負電流後之電刺激皆使細胞面積具有下降之趨勢，依據氧化與還原反應處理可觀察細胞具有不同型態變異。根據電刺激之觀測電化學趨勢與細胞內部之鈣離子螢光偵測分析，我們發現抗藥株於正電流下可使細胞通透性增加且細胞內部鈣離子提升。本研究之電化學之趨勢和其存活率初步了解，抗藥株於正電流之電刺激下使其存活率明顯下降，展示氧化反應對於抗藥株之影響略高於還原反應之電刺激效果，藉此希望未來可以應用且發展於臨床實驗，作為卵巢癌以或相關癌症之電刺激治療基礎。

In this study, we used electrochemical method is used to realize the effect of the electric stimulation, and the effect of the direct current on cancer cell is demonstrated from cell morphology and voltage change. Due to the transparent nature and high conductivity of the indium tin oxide, the conductive glasses coated ITO as a working electrode in three electrode system and culture the cancer cell on the surface. We applied 0.1 mA constant current on cell, we found that whether the cells are resistant to cisplatin, the conductivity of cells to direct current is different, so whether the cells are resistant to cisplatin is different in the electrochemical trends. From the result of survival rate, we found the positive current has more cell deaths than negative current, which indicates that positive current has a greater impact on cisplatin-resistant cells and its survival rate is about 70 %, in comparison to the positive current, the survival rate of cell under negative current which less than 40 %. From the cellular calcium concentration, we found positive current increase cell permeability and increase cellular calcium concentration. As our results, we pointed out the effect of positive and negative current on the cancer cell and cisplatin-resistant cells are different, we show the electrochemical results that the efficiency of positive current and its impact on the cancer cell is greater, indicating that the impact of oxidation reaction is larger than that of reduction reaction on cisplatin-resistant cells. we hope that this application can be used in clinical development one day.

摘要    I
Abstract    II
目錄    III
圖目錄    V
第一章、緒論    1
1-1、前言    1
1-2、研究動機與目的    4
第二章、文獻回顧    6
2-1、卵巢癌之簡介    6
2-2、 卵巢癌之診斷    9
2-3、卵巢癌之治療    14
2-4、卵巢癌細胞對於電化學刺激之研究發想    18
2-4-1、氧化銦錫的材料性質    18
2-4-2、氧化銦錫細胞培養    20
2-4-3、氧化銦錫電極發展及相關電刺激應用    22
第三章、實驗設備與程序    31
3-1、實驗所使用之藥品與實驗設備    31
3-2、細胞培養於氧化銦錫導電玻璃    32
3-3、通電實驗之電化學測試    33
3-4、通電實驗後之相關分析    35
3-4-1、通電實驗結束後之細胞收集    35
3-4-2、通電實驗結束後之存活率測試    35
3-4-3、通電實驗之鈣離通道分析    35
3-4-4、通電實驗之統計分析    36
第四章、實驗結果與討論    38
4-1、細胞培養結果與檢測結果    38
4-1-1、氧化銦錫導電玻璃片導電度測試    38
4-1-2、細胞培養於氧化銦錫導電玻璃片電化學測試    42
4-2、ES-2細胞與其抗藥株通以固定電流通電下之結果    44
4-2-1、ES-2細胞與其抗藥株通以負電流通電下之結果    45
4-2-2、ES-2細胞與其抗藥株通以正電流通電下之結果    49
4-3、A2780細胞與其抗藥株通以固定電流通電下之結果    53
4-3-1、負電流下A2780細胞與其抗藥株通電結果    53
4-3-2、正電流下A2780細胞與其抗藥株通電結果    59
4-4、通電後鈣離子通道的測試    64
4-4-1、ES-2細胞株與抗藥株於固定負電流實驗下細胞內部鈣離子濃度分析    64
4-4-2、ES-2細胞株與抗藥株於固定正電流實驗下細胞內部鈣離子濃度分析    67
4-4-3、A2780細胞株與抗藥株於固定負電流實驗下細胞內部鈣離子濃度分析    70
五、結論與未來工作    75
六、參考文獻    77


                                

1. 衛生福利部統計處 109年死因統計結果分析. https://www.mohw.gov.tw/cp-5017-61533-1.html.
2. institute, N. c. What Is Cancer? https://www.cancer.gov/about-cancer/understanding/what-is-cancer.
3. Yang, Y.; Yang, Y.; Yang, J.; Zhao, X.; Wei, X., Tumor Microenvironment in Ovarian Cancer: Function and Therapeutic Strategy. Frontiers in Cell and Developmental Biology 2020, 8, 758.
4. Frede, J.; Fraser, S. P.; Oskay-Özcelik, G.; Hong, Y.; Braicu, E. I.; Sehouli, J.; Gabra, H.; Djamgoz, M. B., Ovarian cancer: Ion channel and aquaporin expression as novel targets of clinical potential. European journal of cancer 2013, 49 (10), 2331-2344.
5. Giornelli, G. H., Management of relapsed ovarian cancer: a review. Springerplus 2016, 5 (1), 1-11.
6. Jayson, G. C.; Kohn, E. C.; Kitchener, H. C.; Ledermann, J. A., Ovarian cancer. The Lancet 2014, 384 (9951), 1376-1388.
7. Trinidad, C. V.; Tetlow, A. L.; Bantis, L. E.; Godwin, A. K., Reducing ovarian cancer mortality through early detection: approaches using circulating biomarkers. Cancer Prevention Research 2020, 13 (3), 241-252.
8. 財團法人台灣癌症基金會卵巢癌(Ovarian Cancer). https://www.canceraway.org.tw/page.asp?IDno=2215.
9. Vergote, I.; Amant, F.; Kristensen, G.; Ehlen, T.; Reed, N. S.; Casado, A., Primary surgery or neoadjuvant chemotherapy followed by interval debulking surgery in advanced ovarian cancer. European journal of cancer 2011, 47, S88-S92.
10. 陳怡仁醫師, 臺. 婦. 婦女癌症,認識卵巢癌. https://wd.vghtpe.gov.tw/obgy/Fpage.action?fid=3800.
11. 婦癌科醫師卵巢癌(Ovarian Cancer). https://www1.cgmh.org.tw/intr/intr4/c8710/C6/Content/Content4/C6_4_13.htm.
12. Saczko, J.; Kamińska, I.; Kotulska, M.; Bar, J.; Choromańska, A.; Rembiałkowska, N.; Bieżuńska-Kusiak, K.; Rossowska, J.; Nowakowska, D.; Kulbacka, J., Combination of therapy with 5-fluorouracil and cisplatin with electroporation in human ovarian carcinoma model in vitro. Biomedicine & Pharmacotherapy 2014, 68 (5), 573-580.
13. Napotnik, T. B.; Miklavčič, D., In vitro electroporation detection methods–An overview. Bioelectrochemistry 2018, 120, 166-182.
14. Saczko, J.; Pilat, J.; Choromanska, A.; Rembialkowska, N.; Bar, J.; Kaminska, I.; Zalewski, J.; Kulbacka, J., The effectiveness of chemotherapy and electrochemotherapy on ovarian cell lines in vitro. Neoplasma 2016, 63 (3), 450-455.
15. Torre, L. A.; Trabert, B.; DeSantis, C. E.; Miller, K. D.; Samimi, G.; Runowicz, C. D.; Gaudet, M. M.; Jemal, A.; Siegel, R. L., Ovarian cancer statistics, 2018. CA: a cancer journal for clinicians 2018, 68 (4), 284-296.
16. Roett, M. A.; Evans, P., Ovarian cancer: an overview. American family physician 2009, 80 (6), 609-616.
17. Medicine, W. C. Ovarian Cancer. https://weillcornell.org/services/obstetrics-and-gynecology/gynecologic-oncology/conditions-we-treat/ovarian-cancer.
18. Alcázar, J. L.; Utrilla-Layna, J., Malignant Ovarian Tumors (Serous/Mucinous/Endometrioid/Clear Cell Carcinoma): Clinical Setting and Ultrasound Appearance. In Ovarian Neoplasm Imaging, Springer: 2013; pp 229-242.
19. Kossaï, M.; Leary, A.; Scoazec, J.-Y.; Genestie, C., Ovarian cancer: a heterogeneous disease. Pathobiology 2018, 85 (1-2), 41-49.
20. Kurman, R. J.; Shih, I.-M., The Origin and pathogenesis of epithelial ovarian cancer-a proposed unifying theory. The American journal of surgical pathology 2010, 34 (3), 433.
21. Lalwani, N.; Prasad, S. R.; Vikram, R.; Shanbhogue, A. K.; Huettner, P. C.; Fasih, N., Histologic, molecular, and cytogenetic features of ovarian cancers: implications for diagnosis and treatment. Radiographics 2011, 31 (3), 625-646.
22. Meinhold-Heerlein, I.; Fotopoulou, C.; Harter, P.; Kurzeder, C.; Mustea, A.; Wimberger, P.; Hauptmann, S.; Sehouli, J., The new WHO classification of ovarian, fallopian tube, and primary peritoneal cancer and its clinical implications. Archives of gynecology and obstetrics 2016, 293 (4), 695-700.
23. Gershenson, D. M., Current advances in the management of malignant germ cell and sex cord-stromal tumors of the ovary. Gynecologic oncology 2012, 125 (3), 515-517.
24. Maoz, A.; Matsuo, K.; Ciccone, M. A.; Matsuzaki, S.; Klar, M.; Roman, L. D.; Sood, A. K.; Gershenson, D. M., Molecular Pathways and Targeted Therapies for Malignant Ovarian Germ Cell Tumors and Sex Cord–Stromal Tumors: A Contemporary Review. Cancers 2020, 12 (6), 1398.
25. Goff, B. A.; Mandel, L. S.; Drescher, C. W.; Urban, N.; Gough, S.; Schurman, K. M.; Patras, J.; Mahony, B. S.; Andersen, M. R., Development of an ovarian cancer symptom index: possibilities for earlier detection. Cancer 2007, 109 (2), 221-227.
26. 陳渝潔, 高. 卵巢癌的早期篩檢. http://www.doctor.org.tw/file/75-3-2.pdf.
27. Permuth-Wey, J.; Chen, D.-T.; Fulp, W. J.; Yoder, S. J.; Zhang, Y.; Georgeades, C.; Husain, K.; Centeno, B. A.; Magliocco, A. M.; Coppola, D., Plasma microRNAs as novel biomarkers for patients with intraductal papillary mucinous neoplasms of the pancreas. Cancer prevention research 2015, 8 (9), 826-834.
28. Razmi, N.; Hasanzadeh, M., Current advancement on diagnosis of ovarian cancer using biosensing of CA 125 biomarker: Analytical approaches. TrAC Trends in Analytical Chemistry 2018, 108, 1-12.
29. Diaconu, I.; Cristea, C.; Hârceagă, V.; Marrazza, G.; Berindan-Neagoe, I.; Săndulescu, R., Electrochemical immunosensors in breast and ovarian cancer. Clinica Chimica Acta 2013, 425, 128-138.
30. Prevarskaya, N.; Skryma, R.; Shuba, Y., Ion channels and the hallmarks of cancer. Trends in molecular medicine 2010, 16 (3), 107-121.
31. Beebe, S. J.; Sain, N. M.; Ren, W., Induction of cell death mechanisms and apoptosis by nanosecond pulsed electric fields (nsPEFs). Cells 2013, 2 (1), 136-162.
32. Wei, L.; Xiao, A. Y.; Jin, C.; Yang, A.; Lu, Z. Y.; Yu, S. P., Effects of chloride and potassium channel blockers on apoptotic cell shrinkage and apoptosis in cortical neurons. Pflügers Archiv 2004, 448 (3), 325-334.
33. Krabbendam, I. E.; Honrath, B.; Culmsee, C.; Dolga, A. M., Mitochondrial Ca2+-activated K+ channels and their role in cell life and death pathways. Cell Calcium 2018, 69, 101-111.
34. Fraser, S. P.; Ozerlat-Gunduz, I.; Brackenbury, W. J.; Fitzgerald, E. M.; Campbell, T. M.; Coombes, R. C.; Djamgoz, M. B., Regulation of voltage-gated sodium channel expression in cancer: hormones, growth factors and auto-regulation. Philosophical Transactions of the Royal Society B: Biological Sciences 2014, 369 (1638), 20130105.
35. Zeineldin, R.; Muller, C. Y.; Stack, M. S.; Hudson, L. G., Targeting the EGF receptor for ovarian cancer therapy. Journal of oncology 2010, 2010.
36. Chinmoy, K. B., Role of nerve growth factor and FSH receptor in epithelial ovarian cancer. Reproductive biomedicine online 2005, 11 (2), 194-197.
37. Lafky, J. M.; Wilken, J. A.; Baron, A. T.; Maihle, N. J., Clinical implications of the ErbB/epidermal growth factor (EGF) receptor family and its ligands in ovarian cancer. Biochimica et Biophysica Acta (BBA)-Reviews on Cancer 2008, 1785 (2), 232-265.
38. Kondratskyi, A.; Kondratska, K.; Skryma, R.; Prevarskaya, N., Ion channels in the regulation of apoptosis. Biochimica et Biophysica Acta (BBA)-Biomembranes 2015, 1848 (10), 2532-2546.
39. Angus, M.; Ruben, P., Voltage gated sodium channels in cancer and their potential mechanisms of action. Channels 2019, 13 (1), 400-409.
40. Prevarskaya, N.; Skryma, R.; Shuba, Y., Ion channels in cancer: are cancer hallmarks oncochannelopathies? Physiological reviews 2018, 98 (2), 559-621.
41. Kim, J. Y.; Cho, C. H.; Song, H. S., Targeted therapy of ovarian cancer including immune check point inhibitor. The Korean journal of internal medicine 2017, 32 (5), 798.
42. Högberg, T.; Glimelius, B.; Nygren, P., A systematic overview of chemotherapy effects in ovarian cancer. Acta oncologica 2001, 40 (2-3), 340-360.
43. Kolosnjaj-Tabi, J.; Gibot, L.; Fourquaux, I.; Golzio, M.; Rols, M.-P., Electric field-responsive nanoparticles and electric fields: physical, chemical, biological mechanisms and therapeutic prospects. Advanced drug delivery reviews 2019, 138, 56-67.
44. Probst, U.; Fuhrmann, I.; Beyer, L.; Wiggermann, P., Electrochemotherapy as a new modality in interventional oncology: a review. Technology in cancer research & treatment 2018, 17, 1533033818785329.
45. Yang, X.-J.; Li, J.; Sun, C.-X.; Zheng, F.-Y.; Hu, L.-N., The effect of high frequency steep pulsed electric fields on in vitro and in vivo antitumor efficiency of ovarian cancer cell line skov3 and potential use in electrochemotherapy. Journal of Experimental & Clinical Cancer Research 2009, 28 (1), 1-9.
46. Jaatinen, L.; Young, E.; Hyttinen, J.; Vörös, J.; Zambelli, T.; Demkó, L., Quantifying the effect of electric current on cell adhesion studied by single-cell force spectroscopy. Biointerphases 2016, 11 (1), 011004.
47. Veiga, V. F.; Holandino, C.; Rodrigues, M. L.; Capella, M. A.; Menezes, S.; Alviano, C. S., Cellular damage and altered carbohydrate expression in P815 tumor cells induced by direct electric current: an in vitro analysis. Bioelectromagnetics: Journal of the Bioelectromagnetics Society, The Society for Physical Regulation in Biology and Medicine, The European Bioelectromagnetics Association 2000, 21 (8), 597-607.
48. Cury, F. L.; Bhindi, B.; Rocha, J.; Scarlata, E.; El Jurdi, K.; Ladouceur, M.; Beauregard, S.; Vijh, A. K.; Taguchi, Y.; Chevalier, S., Electrochemical red-ox therapy of prostate cancer in nude mice. Bioelectrochemistry 2015, 104, 1-9.
49. Aydın, E. B.; Sezgintürk, M. K., Indium tin oxide (ITO): A promising material in biosensing technology. TrAC Trends in Analytical Chemistry 2017, 97, 309-315.
50. Pallarola, D.; Bochen, A.; Guglielmotti, V.; Oswald, T. A.; Kessler, H.; Spatz, J. P., Highly ordered gold nanopatterned indium tin oxide electrodes for simultaneous optical and electrochemical probing cell interactions. Analytical chemistry 2017, 89 (18), 10054-10062.
51. Moon, S.-H.; Cho, Y.-W.; Shim, H.-E.; Choi, J.-H.; Jung, C.-H.; Hwang, I.-T.; Kang, S.-W., Electrically stimulable indium tin oxide plate for long-term in vitro cardiomyocyte culture. Biomaterials research 2020, 24 (1), 1-8.
52. Guo, M.; Chen, J.; Yun, X.; Chen, K.; Nie, L.; Yao, S., Monitoring of cell growth and assessment of cytotoxicity using electrochemical impedance spectroscopy. Biochimica et Biophysica Acta (BBA)-General Subjects 2006, 1760 (3), 432-439.
53. Love, M. R.; Palee, S.; Chattipakorn, S. C.; Chattipakorn, N., Effects of electrical stimulation on cell proliferation and apoptosis. Journal of cellular physiology 2018, 233 (3), 1860-1876.
54. Khitrin, A.; Khitrin, K.; Model, M., A model for membrane potential and intracellular ion distribution. Chemistry and physics of lipids 2014, 184, 76-81.
55. Vera-Tizatl, C.; Talamás-Rohana, P.; Vera-Hernández, A.; Leija-Salas, L.; Rodríguez-Cuevas, S.; Chávez-Munguía, B.; Vera-Tizatl, A., Cell morphology impact on the set-up of electroporation protocols for in-suspension and adhered breast cancer cells. Electromagnetic Biology and Medicine 2020, 39 (4), 323-339.
56. Turjanski, P.; Olaiz, N.; Abou-Adal, P.; Suarez, C.; Risk, M.; Marshall, G., pH front tracking in the electrochemical treatment (EChT) of tumors: Experiments and simulations. Electrochimica Acta 2009, 54 (26), 6199-6206.
57. Poljsak, B.; Šuput, D.; Milisav, I., Achieving the balance between ROS and antioxidants: when to use the synthetic antioxidants. Oxidative medicine and cellular longevity 2013, 2013.
58. Yao, C.; Mi, Y.; Hu, X.; Li, C.; Sun, C.; Tang, J.; Wu, X. In Experiment and mechanism research of SKOV3 cancer cell apoptosis induced by nanosecond pulsed electric field, 2008 30th Annual International Conference of the IEEE Engineering in Medicine and Biology Society, IEEE: 2008; pp 1044-1047.
59. Frandsen, S. K.; Gissel, H.; Hojman, P.; Tramm, T.; Eriksen, J.; Gehl, J., Direct therapeutic applications of calcium electroporation to effectively induce tumor necrosis. Cancer research 2012, 72 (6), 1336-1341.
60. Frandsen, S. K.; Vissing, M.; Gehl, J., A comprehensive review of calcium electroporation—A novel cancer treatment modality. Cancers 2020, 12 (2), 290.
61. Borst, P.; Evers, R.; Kool, M.; Wijnholds, J., A family of drug transporters: the multidrug resistance-associated proteins. Journal of the National Cancer Institute 2000, 92 (16), 1295-1302.
62. Gao, B.; Yang, F.; Chen, W.; Li, R.; Hu, X.; Liang, Y.; Li, D., Multidrug resistance affects the prognosis of primary epithelial ovarian cancer. Oncology letters 2019, 18 (4), 4262-4269.
63. Hong, J.; Kandasamy, K.; Marimuthu, M.; Choi, C. S.; Kim, S., Electrical cell-substrate impedance sensing as a non-invasive tool for cancer cell study. Analyst 2011, 136 (2), 237-245.
64. Sodek, K. L.; Ringuette, M. J.; Brown, T. J., Compact spheroid formation by ovarian cancer cells is associated with contractile behavior and an invasive phenotype. International journal of cancer 2009, 124 (9), 2060-2070.

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