跳到主要內容

臺灣博碩士論文加值系統

(3.236.110.106) 您好!臺灣時間:2021/07/24 06:06
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

我願授權國圖
: 
twitterline
研究生:宋珮如
研究生(外文):Pei-Ju Sung
論文名稱:多型性神經膠母細胞瘤之放射線媒介基因治療研究
論文名稱(外文):Radiation-mediated gene therapy of glioblastoma multiforme
指導教授:陳富都陳富都引用關係
指導教授(外文):Fu-Du Chen
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:放射醫學科學研究所
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:2004
畢業學年度:92
語文別:中文
論文頁數:89
中文關鍵詞:單純疱疹病毒胸腺嘧啶激脢Cre-loxP重組系統hTERT 啟動子多型性神經膠母細胞瘤基因表現影像
外文關鍵詞:HSVtkCre-loxPhTERT promoterglioblastoma multiformegene expression imaging
相關次數:
  • 被引用被引用:0
  • 點閱點閱:98
  • 評分評分:
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
惡性腦瘤中,多型性神經膠母細胞瘤(GBM-glioblastoma multiforme)具有非常高的侵犯性,並且對傳統放射治療及化學治療抵抗性非常高,故近年來許多研究團隊廣泛的研究惡性腦瘤的治療策略,期望能提高病人的預後。單純疱疹病毒胸腺嘧啶激酶(HSVtk gene)基因與前驅藥物GCV合併之自殺式基因治療為最常被應用在人類中樞神系統腫瘤的治療方式。
本研究探討HSVtk/GCV基因在惡性腦瘤(GBM)細胞的治療成效,並結合放射線提高自殺式基因治療之效果,實驗中結合放射線驅動之分子開關(cre-loxP)和腫瘤特異性啟動子(hTERT promoter),提高活體外治療GBM細胞的效力。
我們設計兩種基因載體建構物,其中一載體建構物包含腫瘤特異性啟動子hTERT和cre recombinase,另一載體則為E4PCMV-lox- luciferase或HSVtk基因,及將此雙基因建構物結合成單一載體建構物,並以lucoferase assay及MTT技術觀察此不同載體建構物之基因表現程度。為了評估活體腫瘤之HSVtk基因表現,我們利用非侵入性之核醫影像(例如:microPET或SPECT),以氟或碘標熾之核酸類似物(例如:18F-FHBG或131I-FIAU)觀察基因表現影像。
依結果顯示,放射線照射後最適合基因表現的劑量點為2格雷,且以照射後48小時基因表現度較高,最適當的腫瘤特異啟動子為全長的hTERT promoter(human telomerase reverse transcriptase -- full length)。體外luciferase活性實驗結果中,結合hTERT腫瘤特異性啟動子的雙載體系統建構物,其luciferase強度比只利用hTERT啟動子之強度高出五倍;且在體外細胞轉染不同載體建構物後對GCV毒性實驗結果中,發現雙載體系統建構物較單一載體系統建構物基因表現強,照射2Gy劑量時,其細胞存活也有比較明顯的差距。當利用TK當報導基因做gamma camera基因表現影像及macroautoradiography時,發現131I-FIAU其藥物之放射活性僅在TK+腫瘤中有明顯積聚現象;在活體治療實驗中,可觀察到單一載體stable clone腫瘤給予GCV後有明顯的生長抑制情形。此證明了在GBM腦瘤細胞中,結合腫瘤特異性啟動子及cre-loxP分子開關可以有效的在活體外及活體內均使基因表現程度增加,並且存在著抑制腫瘤生長的效應,故此研究結果對於未來惡性腦瘤GBM之專一性腫瘤基因治療研究將有很大幫助。
GBM (glioblastoma multiforme) is highly invasive and resistant to conventional radiotherapy and chemotherapy. New therapeutic strategies such as gene therapy must be found in order to improve the prognosis. Herpes simplex virus thymidine kinase gene (HSVtk) transfer together with treatment using the prodrug ganciclovir (GCV) represents the most commonly used suicide gene approach of human central nervous system.
We studied the effects of the herpes simplex virus thymidine kinase/ ganciclovir(HSVtk/GCV)system on GBM cells. In this study, we combined the radiation with suicide gene therapy to enhance the efficacy of treatment. We are currently using a radiation-triggered molecular switching device based on a tumor specific promoter elements(hTERT promoter) and Cre-loxP site-specific recombination system in the treatment of GBM cells in vitro.
We constructed two kinds of plasmids. One plasmid contains a Cre recombinase under the control of hTERT and the other expresses E4PCMV-lox-luciferase/herpes simplex virus thymidine kinase. We also combined the two plasmids into a single plasmid system, which were then tested in vitro to compare the level of gene expression by luciferase assay and cell survival by MTT assay techniques. To monitor HSV-tk gene expression in vivo, we employed noninvasive nuclear imaging modalities (e.g. microPET or SPECT) and a substrate of radioiodinated nucleoside analog(e.g. [131I]-FIAU)to perform gene expression imaging.
Our results demonstrated that radiation dose of 2 Gy, and full length hTERT promoter resulted in highest level of gene expression in vitro. The luciferase and survival data indicated that full length hTERT promoter combined with Cre-loxp system is better than hTERT promoter only. In vitro luciferase activity obtained with hTERT promoter and Cre-loxP double plasmid system was 5-fold higher than that with the hTERT promoter alone. And that in vitro cytotoxic effect of HSVtk/GCV obtained with the hTERT promoter and Cre-loxP double plasmid system is better than single plasmid system. [131I]-FIAU-derived radioactivity distributed only in the area of HSVtk-positive tumors was observed by nuclear imaging and macroauto- radiography. And tumor growth inhibition of single plasmid stable clone was also observed in in vivo tumor models. These findings indicate that the combination of hTERT promoter and Cre-loxP system can enhance the level of gene expression of GBM cells and also exert its anti-tumor effect both in vitro and in vivo. It is concluded that this system is good for future research in tumor specific gene therapy of glioblastoma multiforme.
Badie B., Goh C.S., Klaver J., Herweijer H., Boothman D.A. Combined radiation and p53 gene therapy of malignant glioma cells. Cancer Gene Therapy. 1999;6;2;155-162.
Benazzourz B.R., Laine M., Vicat J.M., Boisseau S., Remy C., Fouilhe N., Thomas F., Nissou M.F., Benabid A.L., Berger F. Therapeutic efficacy of the thymidine kinase/ganciclovior system on large experimental gliomas:a nuclearmagnetic resonance image study. Gene Therapy. 1999;6;1030-1037.
Chen S., Shine H.D., Goodman J.C., Grossman R.G., Woo S.L.C. Gene therapy for brain tumors: regression of experimental gliomas by adenovirus-mediated gene transfer in vivo. Proceedings of the National Academy Sciences. 1994; 91;3054- 3057.
Costantini L.C., Bakowska J.C., Breakefield X.O., Isacson O. Gene therapy in the CNS. Gene Therapy. 2000;7;93-109.
Datta R., Rubin E., Qureshi S., Hallahan D.E., Weichselbaum RR., Kufe D.W. Ionizing radiation activates transcription of the EGR1 gene via CArG elements. Proceedings of the National Academy Sciences. 1992;89;10149-10153.
Datta R., Taneja N., Sukhatma V.P., Qureshi S.A. Reactive oxygen intermediates target CC(A/T)6TT sequences to mediate activation of the early growth response 1 transcription factor gene by ionizing radiation. Proceedings of the National Academy Sciences. 1993;90;2419-2422.
Dharmin N., Ronald V., Menzo H., Cees J.A., Abraham B., Peter S.S. Treatment of malignant gliomas with a replicating adenoviral vector expressing herpes simplex virus- thymidine kinase. Cancer Research.2001;61;8743-8750.
Frank G., Hong L., Chiat C., Paule O., Annelise B.G., Gilles V., Jeannette S., Claudine S., He L., Michel P., Patrice Y. Combined effects of radiotherapy and angiostatin gene therapy in glioma tumor model. Proceedings of the National Academy Sciences. 2000;97;6698-6703.
Freeman S.M., Whartwnby K.A., Freeman J.L., Abbound C.N., Marrogi A.J. In situ use of suiside genes for cancer therapy. Seminars in Oncology.1996; 23;31-45.
Geng L., Walter S., Melian E., Vaughan A.T. Transfection of a vector expression wild-type p53 into cells of two human glioma cell lines enhances radiation toxicity. Radiation Research. 1998; 150;31-37.
Gu J., Andreeff M., Roth J.A., Fang B. hTERT promoter induces tumor-specific Bax gene expression and cell killing in syngenic mouse tumor model and prevents systemic toxicity. Gene Therapy. 2002;9;30-37.
Hallahan D.E., Sukhatme V.P., Sherman M.L., Virudachalam S., Kufe D., Weichselbaum R.R. Protein kinase C mediates X-Ray inducibility of nuclear signal transducers EGR1 and JUN. Proceedings of the National Academy Sciences. 1991; 88: 2156 -2160.
Herschman H.R., MacLaren D.C., Iyer M., Namavari M., Bobinski K., Green L.A., Wu L., Berk A.J., Toyokuni T. Barrio J.R. Non-invasive, quantitative and repetitive imaging of reporter gene expression in living animals, using positron emission tomography. Journal of neuroscience Research. 2000;59;699- 705.
Hiroki N., Masaroshi T., Tadashi M., Iwadate Y., Sakiama S. Treatment of rat experimental brain tumors by herpes simolex virus thymidine kinase gene-transduced allogeneic tumor cells and ganciclovir. Cancer Gene Therapy. 2000;7;947-953
In A.K., Youn J.Y., Sei C.Y., Ihl B. C., Chul S.K., Hee C.K., Chang M.K., Young A.J., Joon K.K., Yong K.H. Potential of adenoviral p53 gene therapy and irradiation for the treatment of malignant gliomas. International Journal of Oncology. 2001;19;1041-1047.
Janouskova O., Sima P., Kunke D. Combined suicide gene and immunostimulatory gene therapy using AAV-mediated gene transfer to HPV-16 transformed mouse cell: Decrease of oncogenicity and induction of protection. International Journal of Oncology. 2003;22;569-577.
Joki T., Nakamura M., Ohno T.Activation of the radiosensitive EGR-1 promoter induces expression of the herpes simplex virus thymidine kinase gene and sensitivity of human glioma cells to ganciclovir. Human Gene Therapy. 1995;6;1507-1513.
Kanazawa T., Mizukami H., Okada T., Hanazono Y., Kume A., Nishino H., Takeuchi K., Kitamura K., Ichimura K., Ozawa K. Suicide gene therapy using AAV-HSVtk/GCV in combination with irradiation results in regression of human head and neck cancer xenografts in nude mice. Gene Therapy. 2003; 10;51-58.
Kianmanesh A.R., Perrin H., Panis Y., Fabre M., Nagy H.J. Houssin D., Klatzman D. A “distant” bystander effect of suicide gene therapy: regression of nontransduced tumors together with a distant transduced tumor. Human Gene Therapy. 1997;8;1807-1814.
Lang F.F., Yung W.K.A., Ra J.U., Floralyn L., Icholas N., Erry H.A.T., Ofilon P.J.T. Enhancement of radio- sensitivity of wild-type human glioma cells by adenovirus- mediated delivery of the p53 gene. Journal of Neurosurgery. 1998;89;125 -132.
Laura Z.R., Paul D.B., Patrick J.M., Mark K.K., Donna S.S. Cytotoxicity and accumulation of ganciclovir triphosphate in bystander cells cocultured with herpes simplex virus type 1 thymidine kinase-expressing human glioblastoma cells. Cancer Research. 1999;59;669-675.
Marples B., Scott S.D., Hendry J.H., Embleton M.J.,Lashford L.S., Margison G.P. Development of synthesis promoters for radiation- mediated gene therapy. Gene Therapy. 2000;7; 511- 517.
Marples B., Greco O., Joiner M.C., Scott SD. Molecular approaches to chemo-radiotherapy. European Journal of Cancer. 2002; 38;231-239.
Richard B.P., Ronald E.W., Claudia L.C., Bridget E.S., Linda M.P. A novel multiply-mutated HSV-1 strain for the treatment of human brain tumors. Human Gene Therapy. 1997;8;533-544.
Rosolen A., Frascella E., Francesco C., Todesco A., Petrone M., Mehtali M., Zacchello F., Zanesco L., Scarpa M. In vitro and in vivo antitumor effect of retrovirus-mediated herped simplex thymidine kinase gene-transfer in human medulloblastoma. Gene Therapy. 1998;5;113-120.
Schmidt N.O., Westphal M., Hagel C., Ergun S., Stavrou D., Rosen E.M., Lamszus K. Levels of vascular endothelial growth factor, hepatocyte growth factor/ scatter factor and basic fibroblast growth factor in human gliomas and their relation to angiogenesis. International Journal of Cancer.1999;84 (1);10-18.
Scott S.D., Marples B., Hendry J.H., Lashford L.S., Embleton M.J., Hunter R.D., Howell A., Margison G.P. A radiation- controlled molecular switch for use in gene therapy of cancer. Gene Therapy. 2000;7;1121-1125.
Selvarangan P., David T.C., Denise R.S., Ronald D.A., Gene P.S. Adeno-associated virus for cancer gene therapy. Cancer Research. 2001; 61;6313 -6321.
Sherman M.L., Datta R., Hallahan D.E., Weichselbaum RR., Kufe D.W. Ionizing radiation regulates expression of the c-jun proto- oncogene. Proceedings of the National Academy Sciences. 1990;87: 5663-5666.
Shinoura N., Chen L., Wani M.A., Young G.K., Jeffrey J.L., Warnick R.E., Simon M., Menon A.G., Wan L.B., Peter J.S. Protein and messenger RNA expression of connexin43 in astrocytomas: implications in brain tumor gene therapy. Journal of Neurosurgery.1996;84;839-846.
Shusei I., Takushi T., Satoshi S., Ichiro Y.,.Tomohiko A., Masamichi H. Development of gene therapy using prostate- specific membrane antigen promoter /enhancer with cre recombinase/loxP system for prostate cancer cells under androgen ablation condition. Japan Journal of Cancer Research. 2002;93;1154-1163.
Stephan A.V., Kelly K.H. Adenoviral gene therapy. The oncologist. 2002;7;46-59.
Stone D., David A., Bolognani F., Lowenstein P.R., Castro M.G. Viral vectors for gene delivery and gene therapy within the endocrine system. Journal of Endocrinology.2000;164;103-118.
Takashi K., Tadashi O., Kazumi N., Toru K., Toshki F., Hiroyuki G., Isao T., Yoshiro T., Tadamitsu K. Application of the cre recombinase/loxP system further enhances anti- tumor effects in cell type-specific gene therapy against carcinoembryonic antigen-producing cancer. Cancer Research. 1999;59;4906-4911.
Tapscott S.J., Miller A.D., Olson J.M., Berger M.S., Groudine M., Spence A.M. Gene therapy of rat 9L gliosarcoma tumors by transduction with selectable genes dose not require drug selection. Proceedings of the National Academy Sciences. 1994; 91;8185-8189.
Tatsuhiro J., Mariko N., and Tsuneya O. Activation of the radiosensitive EGR-1 promoter induces expression of the herpes simplex virus thymidine kinase gene and sensitivity of human glioma cells to ganciclovir. Human Gene Therapy. 1995;6;1507 -1513.
Tjuvajev J.G., Finn R. Noninvasive imaging of herpes simplex virus thymidine kinase gene transfer and expression: a potential method for monitoring clinical gene therapy. Cancer Research. 1996;56;4087-4095.
Tjuvajev J.G., Avril N., Oku T., Sasajima T., Miyagawa T., Joshi R., Safer M., Beattie B., Diresta G., Daghighian F., Augensen F., Koutcher J., Zweit J., Humm J., Larson S.M., Finn R., Blasberg R. Imagimg of herpes simplex virus thymidine kinase gene transfer and expression by positron emission tomography. Cancer Research. 1998;58;4333-4341.
Yoshio S., Shuichi K., Yumi S., Yumi K., Taiki T., Izumu S., Kenichi K. Gene therapy for hepatocellular carcinoma using two recombinant adenovirus vectors with α-fetoprotein promoter and cre/loxP system. Journal of virological methods.2001; 92;5-17.
Yuji N., Eijun N., Makoto I., Hiroyuki N., Shunuchi Y., Masami N. Enhanced efficacy of transcriptionally targeted suicide gene/prodrug therapy for thyroid carcinoma with the cre-loxP system. Cancer Research.1999;59;3049-3052.
Wildner O. In situ use of suicide genes for therapy of brain tumors. Trends in Molecular Medicine.1999;31;421-429.
Wirth T., Zender L., Schulte B., Mundt B., Plentz R., Rudolph K.L., Manns M., Kubicka S.,Kuhnel F. A Telomerase-dependent conditionally replicating adenovirus for selective treatment of cancer. Cancer Research. 2003;63;3181-3188.
Yajing Y., Alexander J., Toyokuni T., Barrio J.R., Gambhir S.S. Quantification of target gene expression by imaging reporter gene expression in living animals. Nature Medicine. 2000;6; 8;933-937.
Zhang C., Zhan K.L. Gene therapy for gastric cancer: A review. World Journal of Gastroenterology. 2003;9;11;2390-2394.
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top
1. 多型性神經膠母細胞瘤基因表現影像之研究
2. [18F]FELT及[18F]FHBG做為HSV1-tk基因造影劑之比較研究暨評估 [18F]FEG及[18F]FPG做為第一型鈉-葡萄糖轉運蛋白示蹤劑之能力
3. 抗登革病毒非結構性蛋白1之C端抗體與自體抗原蛋白質雙硫鍵異構酶在登革致病機制中的角色
4. 動態磁感對比磁振造影對腦血流量之校正
5. 利用piggyBac跳躍子建立多模組分子影像模式偵測活體內小鼠乳癌生長行為
6. 銦-111標誌微脂體化療藥物於大腸直腸癌動物模式之藥物動力學、輻射劑量與療效評估
7. 利用蒙地卡羅方法建立靜態/旋轉強度調控放射治療劑量模擬系統
8. 抗聚乙二醇基因轉植HCC肝癌小鼠之基因造影與利用PEGylated微脂體藥物治療之療效評估
9. 合成放射性碘標幟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
10. 機械臂半自動化合成核醫正子造影劑及其在荷腫瘤動物的生物特性與分子造影研究
11. 放射性氟標誌核苷類似物作為正子斷層造影示蹤劑之研究
12. 嗜菌體展現胜肽系統篩選技術在肺癌細胞特異性之分子診斷與造影之應用
13. 靈芝防治癌症之初期臨床前療效評估
14. 清華大學開放式水池反應器之微劑量學研究-組織等效比例計數器之測試與應用
15. 18F-Fluoroacetat作為正子造影劑之生物特性研究暨放射性氟標記核苷類似物之合成及其作為基因探針於基因造影應用之研究