跳到主要內容

臺灣博碩士論文加值系統

(3.237.38.244) 您好!臺灣時間:2021/07/24 17:17
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

: 
twitterline
研究生:李威德
研究生(外文):Wei-Te Lee
論文名稱:人類腦神經膠質瘤U138MG細胞株經輻射照射後之時程性基因表現
論文名稱(外文):Microarray Analysis of Temporal Gene Responses to Ionizing Radiation on U138 Glioma Cell Line
指導教授:吳國海
指導教授(外文):Frank QH Ngo
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:英文
論文頁數:70
中文關鍵詞:微陣列基因技術U138腦神經膠質瘤游離輻射細胞週期
外文關鍵詞:MicroarrayU138GlioblastomaIonizing radiationCell cycle
相關次數:
  • 被引用被引用:2
  • 點閱點閱:143
  • 評分評分:
  • 下載下載:12
  • 收藏至我的研究室書目清單書目收藏:0
人類腦神經膠質瘤在臨床分級上屬惡性度最高的第四級,目前標準之治療程序為手術後合併放射線或化學治療,然而其成效並不顯著。使得腦神經膠質瘤病人預後較差的其中一個原因可能與其腫瘤細胞本身具有相當的輻射耐受性(radio-resistence)有關。本實驗旨在研究p53基因突變之腦瘤細胞株U138MG之輻射敏感度並藉由基因表現來了解相關之分子機制。我們利用高密度之寡核酸(oligo)微陣列分析來追蹤U138MG細胞經1%致死劑量(10格雷加馬射線)照射下其時程性基因表現,並輔以適當之資料過濾(filtering)以及統計運算以篩選出具有差異性表現之基因群,且結合生物統計之相關資訊以找出具有特殊生物意義之功能性基因群。細胞對加馬射線之存活曲線則以群落形成測試法(colony-forming assay)測得。此外,為了研究受輻射照射後之細胞其細胞週期的改變及細胞凋亡(apoptosis)反應,我們分別在照射後不同時間點收取細胞進行細胞週期的分析以及DNA段裂(DNA ladder)的量測。結果顯示,細胞經10格雷加馬輻射照射後,其細胞週期會延滯在G2/M期,且在照射後36小時達到最大量,約有80%的細胞停留在G2/M期,同時在照射後四天內皆沒有量測到DNA段裂的現象。在微陣列基因表現分析中,集群(clustering)分析可以將差異性表現的基因主要分為兩大類,其中一類是屬於早期表現(early responded)的基因群,其內含之基因大多與調控細胞週期有關,另一類則屬於晚期表現(late responded)的基因群,其表現量之改變則涉及許多生物功能。其中,第一類基因與實驗中G2/M細胞週期檢查點之引發相吻合。值得注意的是,基因表現分析顯示在照射後6到12小時有一組與調控細胞分裂有關之基因群呈現明顯之抑制。綜合上述,我們推測U138MG細胞由於p53蛋白之突變而缺乏細胞凋亡之現象,使得輻射引起之死亡主要來自於輻射引起之增殖死亡(mitotic death),並進而導致細胞無法在長時間之G2期阻滯中進行損傷之修補。此推測可由基因表現分析之結果得到相關之證實。
Being rated as Grade IV, glioblastoma multiforme (GBM) is the most aggressive pathological form of glioma in the central nervous system. The standard way of treating a GBM patient is through surgical resection followed by radio- and/or chemotherapy. However, the median life expectancy after the therapies remains poor. One of the possible causes of the poor prognostic property of GBM can be associated with the relative radio-resistance of the tumor cells. In this thesis, we have employed a U138MG glioma cell line which contains mutated p53 and attempted to understand the radiosensitivity of this cell line via global transcriptional activities. Temporal global gene expression profiling were traced using high-density oligo microarrays, following 10Gy gamma-irradiation, a dose that reduced colony survival of U138 to about 1%. Appropriate filtering and statistical methods were used to search for significantly altered genes and genes of functional interest were identified using bioinformatics sources. Colony-forming assay was used to obtain the survival response curve to gamma irradiation. To investigate the cell-cycle perturbations and apoptotic events as induced by irradiation, we harvested cells at different incubation times post irradiation (PI) for DNA content analysis and DNA laddering assay, respectively. The data indicated that while there was an extended G2/M arrest of the irradiated cells with a maximum 80% accumulation at 36hrs after 10Gy irradiation, there was no evidence of DNA laddering detectable up to 4 days PI. For the microarray gene expression data, our cluster demonstrated that genes could be divided into two categories: one is the early responded genes, of which most of these genes are related to regulation of cell cycle; the other is the late responded genes, most of which are involved in regulation of multiple biological functions. The changes of expression levels of the former appeared to be consistent with the G2/M checkpoint activities. Interestingly, the data revealed that a group of genes whose functions are in the control of mitotic events were down-regulated from 6-12hrs PI. In conclusion, we speculate that the lack of apoptotic events may be explained by the dysfunction of the p53 in U138MG, whereas the radiation-induced cell death would have to come from a catastrophe of mitotic death, following a failure of an attempt to repair damage in an extensive period of G2 arrest. Such a mechanism of radiation-induced cell death would be supported by the results of our gene expression analysis.
1.Kleihues P, Burger PC, Scheithauer BW. The new WHO classification of brain tumours. Brain Pathol. 1993;3:255-268.
2.Fine HA, Dear KB, Loeffler JS, et al. Meta-analysis of radiation therapy with and without adjuvant chemotherapy for malignant gliomas in adults. Cancer. 1993;71:2585-2597.
3.Huncharek M, Muscat J. Treatment of recurrent high grade astrocytoma; results of a systematic review of 1,415 patients. Anticancer Res. 1998; 18:1303-1311.
4.Salcman M, Scholtz H, Kaplan RS, et al. Long-term survival in patients with malignant astrocytoma. Neurosurgery. 1994;34:213-219.
5.Salcman M. Survival in glioblastoma: histoical perspective. Neurosurgery. 1980;7:435-439.
6.Markert J. Giloblastoma multiforme: introduction. Cancer J. 2003; 9:148.
7.Malaise EP, Fertil B, Chavaudra N, et al. Distribution of radiation sensitivities for human tumor cells of specific histological types: comparison of in vitro to in vivo data. Int J Radiat Oncol Biol Phys. 1986;12:617-624.
8.Sehgal A. Molecular changes during the genesis of human gliomas. Semin Surg Oncol. 1998;14:3-12.
9.Levine AJ. p53, the cellular gatekeeper for growth and division. Cell. 1997;88:323-331.
10.Sigal A, Rotter V. Oncogenic mutations of the p53 tumor suppressor: the demons of the guardian of the genome. Cancer Res. 2000;60: 6788-6793.
11.Louis DN. The p53 gene and protein in human brain tumors. J Neuropathol Exp Neurol. 1994;53:11-21.
12.Sidransky D, Mikkelsen T, Schwechheimer K, et al. Clonal expansion of p53 mutant cells is associated with brain tumour progression. Nature. 1992;355:846-847.
13.Hussain SP, Harris CC. Molecular epidermiology of human cancer: contribution of mutation spectra studies of tumor suppressor genes. Cancer Res. 1998;58:4023-4037.
14.Beroud C, Soussi T. p53 gene mutation: software and database. Nucleic Acids Res. 1998;26:200-204.
15.de Vries A, Flores ER, Miranda B, et al. Targeted point mutations of p53 lead to dominant-negative inhibition of wild-type p53 function. Proc Natl Acad Sci. 2002;99:2948-2953.
16.Lee JM, Bernstein A. p53 mutations increase resistance to ionizing radiation. Proc Natl Acad Sci. 1993;90:5742-5746.
17.Li R, Sutphin PD, Schwartz D, et al. Mutant p53 protein expression interferes with p53-independent apoptotic pathways. Oncogene. 1998;16:3269-3277.
18.Fridman JS, Lowe SW. Control of apoptosis by p53. Oncogene. 2003;22:9030-9040.
19.Elledge SJ. Cell cycle checkpoints: preventing an identity crisis. Science. 1996;274:1664-1672.
20.Zhou BB, Elledge SJ. The DNA damage response: putting checkpoints in perspective. Nature. 2000;408:433-439.
21.Iliakis G, Wang Y, Guan J, et al. DNA damage checkpoint control in cells exposed to ionizing radiation. Oncogene. 2003;22:5834-5847.
22.Canman CE, Lim DS, Cimprich KA, et al. Activation of the ATM kinase by ionizing radiation and phosphorylation of p53. Science. 1998;281:1677-1679.
23.Harper JW, Adami GR, Wei N, et al. The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell. 1993;75:805-816.
24.Chan TA, Hermeking H, Lengauer C, et al. 14-3-3Sigma is required to prevent mitotic catastrophe after DNA damage. Nature. 1999; 401:616-620.
25.Peng CY, Graves PR, Thoma RS, et al. Mitotic and G2 checkpoint control: regulation of 14-3-3 protein binding by phosphorylation of Cdc25C on serine-216. Science. 1997;277:1501-1505.
26.Rich T, Allen RL, Wyllie AH. Defying death after DNA damage. Nature. 2000;407:777-783.
27.Kannan K, Amariglio N, Rechavi G, et al. DNA microarrays identification of primary and secondary target genes regulated by p53. Oncogene. 2001;20:2225-2234.
28.Sionov RV, Haupt Y. The cellular response to p53: the decision between life and death. Oncogene. 1999;18:6145-6157.
29.Kuerbitz SJ, Plunkett BS, Walsh WV, et al. Wild-type p53 is a cell cycle checkpoint determinant following irradiation. Proc Natl Acad Sci. 1992;89:7491-7495.
30.Schulze A, Downward J. Navigating gene expression using microarrays - a technology review. Nat Cell Biol. 2001;3:E190-E195.
31.Celis JE, Kruhoffer M, Gromova I, et al. Gene expression profiling: monitoring transcription and translation products using DNA microarrays and proteomics. FEBS Lett. 2000;480:2-16.
32.Pease AC, Solas D, Sullivan EJ, et al. Light-generated oligonucleotide arrays for rapid DNA sequence analysis. Proc Natl Acad Sci. 1994; 91:5022-5026.
33.Hughes TR, Mao M, Jones AR, et al. Expression profiling using microarrays fabricated by an ink-jet oligonucleotide synthesizer. Nat Biotechnol. 2001;19:342-347.
34.Blanchard AP, Kaiser RJ, Hood LE. High-density oligonucleotide arrays. Biosens Bioelectron. 1996;11:687-690.
35.Ross DT, Scherf U, Eisen MB, et al. Systematic variation in gene expression patterns in human cancer cell lines. Nat Genet. 2000; 24:227-235.
36.Stankovic T, Hubank M, Cronin D, et al. Microarray analysis reveals that TP53- and ATM-mutant B-CLLs share a defect in activating proapoptotic responses after DNA damage but are distinguished by major differences in activating prosurvival responses. Blood. 2004;103:291-300.
37.Elledge SJ, Davis RW, Lockhart DJ. Transcriptional regulation and function during the human cell cycle. Nat Genet. 2001;27:48-54.
38.Tada M, Matsumoto R, Iggo RD, et al. Selectively sensitivity to radiation of cerebral glioblastomas harboring p53 mutations. Cancer Res. 1998;58:1793-1797.
39.Asaoka K, Tada M, Sawamura Y, et al. Dependence of efficient adenoviral gene delivery in malignant glioma cells on the expression levels of the Coxsackievirus and adenovirus receptor. J Neurosurg. 2000;92:1002-1008.
40.Hsiao YY. The effects of radiation on p53-mutated glioma cells using cDNA microarray technique. Master thesis 2003. Institute of Radiological Sciences, National Yang-Ming University.
41.Gong J, Traganos F, Darzynkiewicz Z. A selective procedure for DNA extraction from apoptotic cells applicable for gel electrophoresis and flow cytometry. Anal Biochem. 1994;218:314-319.
42.Draghici S, Khatri P, Bhavsar P, et al. Onto-Tools, the toolkit of the modern biologist: Onto-Express, Onto-Compare, Onto-Design and Onto-Translate. Nucleic Acids Res. 2003;31:3775-3781.
43.Zhang B, Schmoyer D, Kirov S, et al. GOTree Machine (GOTM): a web-based platform for interpreting sets of interesting genes using Gene Ontology hierarchies. BMC Bioinformatics. 2004;5:16.
44.Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001;25:402-408.
45.Bustin SA. Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol. 2000;25:169-193.
46.Khodarev NN, Sokolova IA, Vaughan AT. Mechanisms of induction of apoptotic DNA fragmentation. Int J Radiat Biol. 1998;73:455-467.
47.Kiechle FL, Zhang X. Apoptosis: biochemical aspects and clinical implications. Clin Chim Acta. 2002;326:27-45.
48.Yao KC, Komata T, Kondo Y, et al. Molecular response of human glioblastoma multiforme cells to ionizing radiation: cell cycle arrest, modulation of the expression of cyclin-dependent kinase inhibitors, and autophagy. J Neurosurg. 2003;98:378-384.
49.Yount GL, Haas-Kogan DA, Vidair CA, et al. Cell cycle synchrony unmasks the influence of p53 function on radiosensitivity of human glioblastoma cells. Cancer Res. 1996;56:500-506.
50.Dudoit S, Yang YH, Callow MJ, et al. Statistical methods for identifying differentially expressed genes in replicated cDNA microarray experiments. Statistica Sinica. 2002;12:111-139.
51.Khatri P, Draghici S, Ostermeier GC, et al. Profiling gene expression using onto-express. Genomics. 2002;79:266-270.
52.Bailis JM, Forsburg SL. MCM proteins: DNA damage, mutagenesis and repair. Curr Opin Genet Dev. 2004;14:17-21.
53.Reed JC. Mechanisms of apoptosis. Am J Pathol. 2000;157:1415-1430.
54.Marsters SA, Sheridan JP, Donahue CJ, et al. Apo-3, a new member of the tumor necrosis factor receptor family, contains a death domain and activates apoptosis and NF-kappa B. Curr Biol. 1996;6:1669-1676.
55.Innocente SA, Abrahamson JL, Cogswell JP, et al. p53 regulates a G2 checkpoint through cyclin B1. Proc Natl Acad Sci. 1999;96:2147-2152.
56.Chung E, Chen RH. Phosphorylation of Cdc20 is required for its inhibition by the spindle checkpoint. Nat Cell Biol. 2003;5:748-753.
57.Chen IT, Smith ML, O''Connor PM, et al. Direct interaction of Gadd45 with PCNA and evidence for competitive interaction of Gadd45 and p21Waf1/Cip1 with PCNA. Oncogene. 1995;11:1931-1937.
58.Yuen T, Wurmbach E, Pfeffer RL, et al. Accuracy and calibration of commercial oligonucleotide and custom cDNA microarrays. Nucleic Acids Res. 2002;30:e48.
59.Smits VA, Medema RH. Checking out the G(2)/M transition. Biochim Biophys Acta. 2001;1519:1-12.
60.Krause K, Wasner M, Reinhard W, et al. The tumour suppressor protein p53 can repress transcription of cyclin B. Nucleic Acids Res. 2000;28:4410-4418.
61.Straight AF. Cell cycle: checkpoint proteins and kinetochores. Curr Biol. 1997;7:R613-R616.
62.Amon A. The spindle checkpoint. Curr Opin Genet Dev. 1999;9:69-75.
63.Burke DJ. Complexity in the spindle checkpoint. Curr Opin Genet Dev. 2000;10:26-31.
64.Kufer TA, Sillje HH, Korner R, et al. Human TPX2 is required for targeting Aurora-A kinase to the spindle. J Cell Biol. 2002;158:617-623.
65.Hirota T, Kunitoku N, Sasayama T, et al. Aurora-A and an interacting activator, the LIM protein Ajuba, are required for mitotic commitment in human cells. Cell. 2003;114:585-598.
66.Pereira G, Schiebel E. Separase regulates INCENP-Aurora B anaphase spindle function through Cdc14. Science. 2003;302:2120-2124.
67.Chen Y, Riley DJ, Chen PL, et al. HEC, a novel nuclear protein rich in leucine heptad repeats specifically involved in mitosis. Mol Cell Biol. 1997;17:6049-6056.
68.Martin-Lluesma S, Stucke VM, Nigg EA. Role of Hec1 in spindle checkpoint signaling and kinetochore recruitment of Mad1/Mad2. Science. 2002;297:2267-2270.
69.DeLuca JG, Howell BJ, Canman JC, et al. Nuf2 and Hec1 are required for retention of the checkpoint proteins Mad1 and Mad2 to kinetochores. Curr Biol. 2003;13:2103-2109.
70.Fei P, El-Deiry WS. P53 and radiation responses. Oncogene. 2003;22: 5774-5783.
71.Yasumoto J, Imai Y, Takahashi A, et al. Analysis of apoptosis-related gene expression after X-ray irradiation in human tongue squamous cell carcinoma cells harboring wild-type or mutated p53 gene. J Radiat Res. 2003;44:41-45.
72.Erenpreisa J, Cragg MS. Mitotic death: a mechanism of survival? A review. Cancer Cell Int. 2001;1:1-7.
73.Castedo M, Perfettini JL, Roumier T, et al. Cell death by mitotic catastrophe: a molecular definition. Oncogene. 2004;23:2825-2837.
74.Valerie K, Povirk LF. Regulation and mechanisms of mammalian double-strand break repair. Oncogene. 2003;22:5792-5812.
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top
1. 許濱松 (民84) 英美公務員政治中立的研究-兼論我國公務員政治中立應有之作法 人事月刊20(4) 頁11-17。
2. 劉清明 (民91) 內部稽核的新發展 內部稽核季刊 頁67~ 74。
3. 劉秀鈴 (民87) 漫談內部控制制度法令史 聯捷會計25 頁16-18。
4. 黃琬玲 (民90) 內部稽核人員的發展 內部稽核35 頁65-68。
5. 張信一 (民90) 如何適當扮演政府之內部稽核角色--從主計人員之職責與專業談起 內部稽核35 頁30-34。
6. 馬秀如 (民88) 內部控制、內部稽核及內部審核範圍之探討 主計月報521 頁26-33。
7. 馬秀如 (民87) 回應爭議與建議再探內部控制的迷思與辨正 會計研究月刊149頁63-80。
8. 柯承恩、賴森本 (民91) 推動績效導向之審計制度 研考雙月刊26(5) 頁59-65。
9. 林源慶 (民87) 從內部控制觀念談我國政府財務活動之非直線監督功能與改進之道 審計季刊18(4) 頁108-112。
10. 吳弘仁 林文隆 (民91) 如何營造高效能之內稽團隊 內部稽核39 頁45-46。
11. 杜文貴 (民89) 內稽與ISO9000之整合 內部稽核季刊33期 頁1~139。
12. 朱楠賢 (民80) 形塑公共組織為學習型組織之初探 人事月刊23(2) 頁60-68。
13. 蔡兆陽 (民88) 邁向政府採購制度的新紀元-政府採購法的實行 主計月報526 頁21-22。
14. 賴文恭 (民80) 內部審核實務上之作業重點探討 今日會計49 頁32~44。
15. 謝俊義 (民87) 新制度論與公共行政:整合性觀點 中國行政評論7 (4)。