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研究生:吳學鵬
研究生(外文):Hsueh-Peng Wu
論文名稱:發展結合IUdR和穿透式特性X光照射的腫瘤鄂惹電子治療
論文名稱(外文):Development of combined IUdR and transmission enriched characteristic X-ray irradiation for cancer Auger electron therapy
指導教授:劉仁賢劉仁賢引用關係
指導教授(外文):Ren-Shyan Liu
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生物醫學影像暨放射科學系
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2014
畢業學年度:103
語文別:中文
論文頁數:70
中文關鍵詞:五碘去氧尿嘧啶鄂惹電子彗星試驗合併治療鈷六十細胞群落形成法鄂惹治療
外文關鍵詞:IUdRAuger electronNG4TL4Comet assayγ-H2AXCo-60Colony formation assayFVB/NμSPECT/CT
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放射線治療一直是癌症治療的重要工具之一,為了提高治療效果,在臨床上也常合併藥物化學治療或搭配輻射增敏劑。輻射增敏劑作用目的在於提升腫瘤對於放射線的敏感度,如此可以避免使用過多的輻射照射,降低正常組織的輻射傷害。五碘去氧尿嘧啶(5-iododeoxyuridine, IUdR)為鹵素化嘧啶(halogenated pyrimidine),是輻射增敏劑的一種,能在細胞DNA合成時併入DNA中,近年來許多科學團隊嘗試利用此藥物搭配碘原子K層電子束縛能的單能X光照射,產生鄂惹電子 (Auger electron)來進行癌症治療。鄂惹電子線性能量轉移 (linear energy transfer, LET)高達100 keV/μm,能嚴重破壞DNA造成細胞死亡,此種治療方式預期能達到比輻射增敏還要更好的腫瘤毒殺效果。
本研究透過與生技公司合作,利用其發展中的穿透式特性X光機,搭配鉭金屬(Tantalum, Ta)為靶材,提高33keV左右能量X光的比例,來進行合併IUdR的腫瘤鄂惹電子治療的測試。此X光機在不同輸出條件下可產生10%,20%及30% 33-40keV的X光。實驗採用NG4TL4小鼠纖維肉瘤來進行所有活體外以及動物腫瘤實驗。實驗包含使用三種不同照射條件以及鈷六十,合併IUdR給予NG4TL4細胞治療,治療後以群落形成法觀察存活狀況,以彗星試驗以及磷酸化 γ-H2AX 免疫螢光染色來偵測DNA斷裂情況。活體方面,在動物腫瘤形成後也以碘123所標誌的IUdR進行μSPECT/CT造影,觀察藥物在腫瘤的代謝情形。最後給予不同條件的治療觀察腫瘤治療情況。
實驗結果顯示,NG4TL4以越高濃度(1-16μg/ ml)的IUdR,DNA中thymidine的替換率會隨之增高(20%~60%)。細胞處理7.8μg IUdR 24小時後以不同條件照射,相較於單獨放射線照射或單獨IUdR處理,都有較高的細胞致死效果以及DNA斷裂情況顯著的提高。相較於IUdR合併鈷六十,IUdR合併穿透式X光機照射可產生較佳的細胞致死率以及較高的DNA破壞程度,顯見IUdR的鄂惹電子治療效果優於單純輻射增敏效果。再者,三種產生不同比例33-40keV的照射條件搭配IUdR處理的實驗結果顯示,比例最高的(30%)條件可造成最嚴重的DNA斷裂結果。活體實驗方面,在建立NG4TL4腫瘤後,小鼠以123IUdR注射腫瘤後進行μSPECT/CT造影追蹤IUdR的代謝情形。治療實驗結果顯示,搭配IUdR腫瘤注射與4Gy的NanoRay照射,相較於單純給予放射線照射或單獨IUdR的治療,可以成功抑制腫瘤的生長,延長小鼠存活。
本研究初步驗證了IUdR搭配提高~33keV能量光子比例的X光照射,對腫瘤進行鄂惹電子治療。除了提高DNA破壞率,降低細胞存活率,也在活體實驗中證實腫瘤的控制率。此治療模式對於未來臨床癌症治療極具潛力,值得進一步發展。

Radiotherapy is one of the important tools of cancer therapy. In order to increase the therapeutic effect, its use often combines with chemotherapy or radiosensitizers in clinical practice. Radiosensitizers aims to increase the radiosensitivity of tumor and the irradiation dose delivered to tumor can consequently be lowered, also the radiation damage to normal tissues. 5-iododeoxyuridine (IUdR) is one type of radiosensitizers categorized as halogenated pyrimidine which can incorporates into nascent DNA during DNA synthesis. Recently, many investigators attempt to treat cancer with IUdR combined irradiation with energy of the K-edge of iodidine to generate Auger electrons for cancer therapy. The lnear energy transfer (LET) of an Auger electron can be as high as 100 keV/μm which is capable of inducing an enormous DNA damage leading to cell death.
This study is conducted via cooperation with a biotech company and use their under-developing transmission X-ray tube equipped with Tantalum target (the NanoRay) which generates 33-40 keV- enriched photons dependent on the target element. With different settings, the NanoRay produces X-ray with different propotion (10%,20% and 30%) of 33-40 keV- enriched photons. Irradiation using Co-60 was included in the experiment as comparison. NG4TL4 fibrosarcoma cell line was used in this study. After treatment of IUdR combined with X-ray of 3 setings or Co-60 irradiation, cell survival was measured using colony formation assay, DNA damage was assayed by comet assay and γ-H2AX immunofluorescent staining. Metabolism of tumorally injected 123IUdR was assessed μSPECT/CT imaging. Auger electron therapy of tumor was carried out by tumoral IUdR injection followed by NanoRay irradiation.
When cells incubated with different concentration of IUdR, thymidine replacement rate increased (20 to 60%) as the IUdR concentration increased (1 to 16 μg/ ml). 10-15 nmole IUdR was detected in 106 cells. IUdR+NanoRay irradiation resulted in higher cell killing effect and DNA damage than irradiation alone or IUdR treatment alone. Compared with IUdR+Co-60 irradiation, IUdR+NanoRay showed better cell killing effect and higher DNA damage, which indicates better therapeutic potential of Auger effect than radiosensitization. Further, when comparing the therapeutic results from IUdR+NanoRay with 3 settings, NanoRay with highest propotional 33-40 keV (30%) induced most severe DNA damage. For in vivo tumor treatment, IUdR+NanoRay irradiation signidficantly inhibited tumor growth as compared to IUdR treatment or NanoRay irradiation alone.
This study preliminarily validated the therapeutic effect of Auger electron therapy for cancer by combining IUdR treatment and X-irradiation with enriched 33-40 keV photons. This treatment regimen not only increased the DNA damage, cell killing but also inhibited tumor growth. In conclusion, this treatment model possesses high potential to be developed for future clinical cancer therapy.

目錄

中文摘要…………………………………………………………………I
英文摘要……………………………………………………..…………IV
目錄………………………………………………..……………………VI
圖目錄………………………………………..……….…………………X
表目錄……………………………………….………………………..XIII
1. 緒言…………………………………………………………..……...1
1.1 輻射偵敏劑………………………………………………..1
1.2 IUdR的特性……………………………………………….2
1.3 鄂惹電子(Auger electron)的特性………………….……..3
1.4 同步輻射…………………………………………………..6
1.5 穿透式X光管……………………………………………..7
2. 實驗目的………………………………………………………….....9
3. 材料與方法………………………………………………………...10
3.1材料…..…………………………………..………………...10
3.1.1 實驗動物…………………………………………..10
3.1.2 細胞株……………………………………………..10
3.1.3 細胞培養與染色材料……………………………..10
3.2化學藥劑…………………………………………………...11
3.3材料配製……………………………………………...……13
3.3.1 1x MEM培養液配製…………………………….13
3.3.2 1x Phosphate buffer saline solution(PBS)配製…..13
3.3.3 0.125%的胰蛋白酶 (Trypsin)…………..…….…13
3.3.4 DNA萃取之細胞裂解液配製………..………….14
3.3.5 電泳液配製…………………………………...….14
3.3.6 中和溶液配製……………………………………14
3.3.7 PBST配製………………………………………..14
3.3.8 0.3%的Triton X-100…………………..…..……..15
3.3.9 Blocking buffer(10 ml)…………………………...15
3.3.10 Antibody dilution buffer (5 ml)…………………15
3.3.11 70%酒精(10 ml)………..……………………….15
3.4實驗方法…………………………………….……………..16
3.4.1 細胞培養………………………….…………….16
3.4.2 腫瘤及動物……………………………………..16
3.4.3 照射條件………………………..………………17
3.4.4 彗星試驗(Comet assay)………………………...18
3.4.5 磷酸化 γ-H2AX 免疫螢光染色………….……19
3.4.6 細胞計數法(Cell count)………………………20
3.4.7 細胞群落形成法(Colony formation assay).....….21
3.4.8 IUdR濃度存活曲線………………...…………..22
3.4.9 IUdR 攝取實驗………………………..………..22
3.4.10 Genomic DNA萃取……………………………22
3.4.11 NG4TL4 纖維肉瘤活體內腫瘤生長曲線…....23
3.4.12 腫瘤治療……………………..……………......23
3.4.13 以μSPECT/CT影像量測123IUdR腫瘤注射後活體內代謝……24
3.4.14 統計方法………………………………………24
4. 結果……………………..………………………………...………..26
4.1 IUdR對細胞毒性測試……………………...…………....26
4.2 穿透式特性X光機(NanoRay)之X光輸出..………..…..26
4.3 IUdR 併入NG4TL4細胞DNA分析……………………27
4.4 IUdR搭配NanoRay照射對NG4TL4細胞DNA斷裂的影響………28
4.5 以免疫螢光染色偵測γ-H2AX磷酸化……..…....……...29
4.6 IUdR搭配NanoRay照射對NG4TL4細胞存活率的影響…………31
4.7 活體腫瘤治療………………....……………………........33
4.8 活體內IUdR濃度存活曲線和照射NanoRay劑量測試..33
4.9 μSPECT/ CT影像……………………………………......34
4.10 腫瘤生長延遲 (growth delay) 試驗………..…..……..34
5. 討論………………………………………………………………...36
6. 結論………………………………………………………….…..…41
7. 參考文獻…………………………………………………..…...…..42
8. 圖表……………………………………….………………..………46

圖目錄

圖一、特製架台(卡通圖)……………………………………………….46
圖二、特製架台(實際)…………………………………………………47
圖三、彗星試驗的五層膠………………………………………………48
圖四、磷酸化 γ-H2AX 免疫螢光染色的操作流程…………………48
圖五、細胞群落形成法的操作流程……………………………..…..…49
圖六、NG4TL4細胞的IUdR濃度曲線……………….………………..50
圖七、不同條件的NanoRay…………………………………………….51
圖八、不同時間磷酸化 γ-H2AX 免疫螢光染色測試………………52
圖九、單獨照射放射線和給予IUdR經24小時再照射放射線在彗星
試驗(Comet assay)中彼此的差異………………………...……53
圖十、給予IUdR經24小時再照射各比例33~40keV的NanoRay和
給予IUdR經24小時再照射鈷六十在彗星試驗(Comet assay)
中彼此的差異………………………………………………..…54
圖十一、給予IUdR經24小時合併四種條件處理後細胞DNA斷裂情
況在彗星試驗(Comet assay)中各劑量彼此間的差異..….……55
圖十二、單獨照射放射線和給予IUdR經24小時再照射放射線在
磷酸化 γ-H2AX 免疫螢光染色中彼此的差異…………..…..56
圖十三、給予IUdR經24小時再照射各比例33~40keV的NanoRay
和給予IUdR經24小時再照射鈷六十在磷酸化 γ-H2AX免疫
螢光染色中彼此的差異…..…………..………………………. 57
圖十四、給予IUdR經24小時合併四種條件處理後細胞DNA損傷情
況在磷酸化 γ-H2AX免疫螢光染色中各劑量彼此間的差異..58
圖十五、單獨照射放射線和給予IUdR經24小時再照射放射線在細
胞群落形成法(Colony formation assay)中彼此的差異…….....59
圖十六、給予IUdR經24小時再照射各比例33~40keV的NanoRay
和給予IUdR經24小時再照射鈷六十在細胞群落形成法
(Colony formation assay)中彼此的差異………...……………..60
圖十七、給予IUdR經24小時合併四種條件處理後細胞毒殺效果在
細胞群落形成法(Colony formation assay)中各劑量彼此間的差
異………………………………………………………………..63
圖十八、IUdR對thymidine的置換率……………………….……...…..64
圖十九、進入細胞和進入細胞中DNA的IUdR比較………………….64
圖二十、NG4TL4纖維肉瘤活體內腫瘤生長曲線……………….……65
圖二十一、建立活體內IUdR濃度曲線……………………………..…66
圖二十二、建立活體內照射放射線劑量測試…………………………66
圖二十三、腫瘤生長延遲(growth delay)試驗…………………..……..67
圖二十四、μSPECT/ CT 影像………………….……………………....68
圖二十五、μSPECT/ CT 腫瘤肌肉組織比…………………..….……..68
圖二十六、新舊穿透式X光管的比較……………………………..…..69

表目錄

表一、活體內IUdR濃度曲線和照射放射線劑量測試各天數各組小鼠的數目……70
表二、腫瘤生長延遲(growth delay)試驗各天數各組小鼠的數目……70

1. Szybalski W and Djordjevic B. Radiation sensitivity of chemically modified human cells. Genetics 1959;44:540-541.
2. Komarnicky LT, Phillips TL, Martz K, Asbell S, Isaacson S, Urtasun R. A randomized phase III protocol for the evaluation of misonidazole combined with radiation in the treatment of patients with brain metastases (RTOG-7916). Int J Radiat Oncol Biol Phys. 1991;20:53-58.
3. Saunder MI, Dische S, Anderson P, Flockhart IR. The neurotoxicity of misonidazole and its relationship to does, half-life and concentration in the serum. Br J Cancer Suppl. 1978;3:268-270.
4. Philip PA, Bagshawe KD, Searle F, Green AJ, Begent RH, Newlands ES, Rustin GJ, Adam T. In vivo uptake of 131I-5-iodo-2-deoxyuridine by malignant tumours in man. Br J Cancer. 1991;63:134-135.
5. Kinsella TJ, Dobson PP, Mitchell JB, Fornace AJ Jr. Enhancement of X ray induced DNA damage by pre-treatment with halogenated pyrimidine analogs. Int J Radiat Oncol Biol Phys. 1987;13:733-739.
6. Prusoff WF. Synthesis and biological activities of iododeoxyuridine, an analog of thymidine. Biochim Biophys Acta. 1959;32:295-296.
7. Chang AE, Collins JM, Speth PA, Smith R, Rowland JB, Walton L, Begley MG, Glatstein E, Kinsella TJ. A phase I study of intraarterial iododeoxyuridine in patients with colorectal liver metastases. J Clin Oncol. 1989;7:662-668.
8. Kalpan HS, Simth KC, Tomlin P. Radiosensitization of E.coli by purine and purimidine analogues incorprorated into deoxyribonucleic acid. Nature. 1961;190:794-796.
9. Steel GG,Wheldon TE. Targeted Radiotherapy. Basic Clinical Radiobiology (2nd). New York: Oxford Uni. Press, Inc., 1997,pp: 224-233.
10. O'Donoghue JA, Wheldon TE. Targeted radiotherapy using Auger electron emitters. Phys Med Biol. 1996;41:1973-1992.
11. Erikson RL, Szybalski W. Molecular radiobiology of human cell lines III. Radiation-sensitizing properties of 5-iododeoxyuridine.Cancer Research. 1963;23:122-130.
12. Lawrence TS, Davis MA, Maybaum J, Stetson PL, Ensminger WD.The dependence of halogenated pyrimidine incorporation and radiosensitization on the duration of drug exposure. Int J Radiat Oncol Biol Phys. 1990;18:1393-1398.
13. Commerford SL, Joel DD. Iododeoxyuridine administered to mice is de-iodinated and incorporated into DNA primarily as thymidylate. Biochem Biophys Res Commun. 1979;86:112-118.
14. Klecker RW, Jenkins JF, Kinsella TJ, Fine RL, Strong JM, Collins JM. Clinical pharmacology of 5-iodo-2'-deoxyuridine and 5-iodouracil and endogenous pyrimidine modulation. Clin Pharmacol Ther. 1985;38:45-51.
15. Kassis AI. 5-123I/125I-iodo-2'-deoxyurid ine for cancer diagnosis and therapy. J Nucl Med Allied Sci. 1990;34:299-303.
16. Cook JA, Glass J, Lebovics R, Bobo H, Pass H, DeLaney TF, Oldfield EH, Mitchell JB, Glatstein E, Goffman TE. Measurement of thymidine replacement in patients with high grade gliomas, head and neck tumors, and high grade sa mlomas after continuous intravenous infusions of 5-iododeoxyuridine. Cancer Res. 1992;52:719-725.
17. Adelstein SJ, Kassis AI, Bodei L, Mariani G. Radiotoxicity of iodine-125 and other auger-electron-emitting radionuclides: background to therapy. Cancer Biother Radiopharm. 2003; 18:301-316.
18. LeMotte PK, Little JB. DNA damage induced in human diploid cells by decay of incorporated radionuclides. Cancer Res. 1984; 44:1337-1342.
19. Makrigiorgos GM, Berman RM, Baranowska-Kortylewicz J, Bump E, Humm JL, Adelstein SJ, Kassis AI. DNA damage produced in V79 cells by DNA-incorporated iodine-123: a comparison with iodine-125. Radiat Res. 1992;129:309-314.
20. Narra VR, Howell RW, Harapanhalli RS, Sastry KS, Rao DV. Radiotoxicity of some iodine-123, iodine-125 and iodine-131-labeled compounds in mouse testes: implications for radiopharmaceutical design. J Nucl Med. 1992;33:2196-2201.
21. Howell RW, Goddu SM, Bishayee A, Rao DV.Radioprotection against lethal damage caused by chronic irradiation with radionuclides in vitro. Radiat Res. 1998;150:391-399.
22. Dugas JP, Varnes ME, Sajo E, Welch CE, Ham K, Hogstrom KR.Dependence of cell survival on iododeoxyuridine concentration in 35-keV photon-activated auger electron radiotherapy. Int J Radiat Oncol Biol Phys. 2011;79:255-261.
23. Biston MC, Joubert A, Charvet AM, Balosso J, Foray N. In vitro and in vivo optimization of an anti-glioma modality based on synchrotron X-ray photoactivation of platinated drugs. Radiat Res. 2009 ;172:348-358.
24. Gastaldo J, Bencokova Z, Massart C, Joubert A, Balosso J, Charvet AM, Foray N. Specific molecular and cellular events induced by irradiated X-ray photoactivatable drugs raise the problem of co-toxicities: particular consequences for anti-cancer synchrotron therapy. J Synchrotron Radiat. 2011;18:456-463.
25. Corde S, Joubert A, Adam JF, Charvet AM, Le Bas JF, Estève F, Elleaume H, Balosso J. Synchrotron radiation-based experimental determination of the optimal energy for cell radiotoxicity enhancement following photoelectric effect on stable iodinated compounds. Br J Cancer. 2004;91:544-551.
26. Rousseau J, Boudou C, Estève F, Elleaume H. Convection-enhanced delivery of an iodine tracer into rat brain for synchrotron stereotactic radiotherapy. Int J Radiat Oncol Biol Phys. 2007;68:943-951.
27. Schulz CA, Mehta MP, Badie B, McGinn CJ, Robins HI, Hayes L, Chappell R, Volkman J, Binger K, Arzoomanian R, Simon K, Alberti D, Feierabend C, Tutsch KD, Kunugi KA, Wilding G, Kinsella TJ. Continuous 28-day iododeoxyuridine infusion and hyperfractionated accelerated radiotherapy for malignant glioma: a phase I clinical study. Int J Radiat Oncol Biol Phys. 2004;59:1107-1115.
28. Rousseau J, Barth RF, Fernandez M, Adam JF, Balosso J, Estève F, Elleaume H. Efficacy of intracerebral delivery of cisplatin in combination with photon irradiation for treatment of brain tumors. J Neurooncol. 2010;98:287-295.
29. Rousseau J, Boudou C, Barth RF, Balosso J, Estève F, Elleaume H. Enhanced survival and cure of F98 glioma-bearing rats following intracerebral delivery of carboplatin in combination with photon irradiation. Clin Cancer Res. 2007;13:5195-5201.
30. Biston MC, Joubert A, Adam JF, Elleaume H, Bohic S, Charvet AM, Estève F, Foray N, Balosso J. Cure of Fisher rats bearing radioresistant F98 glioma treated with cis-platinum and irradiated with monochromatic synchrotron X-rays. Cancer Res. 2004;64:2317-2323.
31. Corde S, Balosso J, Elleaume H, Renier M, Joubert A, Biston MC, Adam JF, Charvet AM, Brochard T, Le Bas JF, Estève F, Foray N. Synchrotron photoactivation of cisplatin elicits an extra number of DNA breaks that stimulate RAD51-mediated repair pathways. Cancer Res. 2003;63:3221-3227.
32. Kobayashi K, Usami N, Porcel E, Lacombe S, Le Sech C. Enhancement of radiation effect by heavy elements. Mutat Res. 2010 ;704:123-131.
33. Ricard C, Fernandez M, Requardt H, Wion D, Vial JC, Segebarth C, van der Sanden B. Synergistic effect of cisplatin and synchrotron irradiation on F98 gliomas growing in nude mice. J Synchrotron Radiat. 2013;20:777-784.
34. 遊潮,劉曉東,吳波,蔡博文. 125IUdR治療C6腦膠質瘤的實驗研究,四川大學學報(醫學版)2004.
35. 郭容妏,雙元放射增敏劑合併放射線對KHT 肉瘤療效之研究。國立陽明大學放射醫學科學研究所碩士論文。2002
36. 黃廣良,*IUdR於肝腫瘤細胞核之輻射劑量評估-於荷肝腫瘤小鼠之肝動脈治療模式。國立陽明大學放射醫學科學研究所碩士論文。2002
37. 郭偉迪,合成放射性碘標誌之胞嘧啶核苷類似物作為腫瘤增生及HSV1-tk基因表現造影劑之研究。國立陽明大學放射醫學科學研究所碩士論文。2009
38. 方信于,兩種輻射增敏藥物 (IUdR 及MISO)在長有KHT肉瘤C3H 小鼠之體內分佈。國立陽明大學放射醫學科學研究所碩士論文。2001

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