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研究生(外文):Wei-Te Lee
論文名稱(外文):Microarray Analysis of Temporal Gene Responses to Ionizing Radiation on U138 Glioma Cell Line
指導教授(外文):Frank QH Ngo
外文關鍵詞:MicroarrayU138GlioblastomaIonizing radiationCell cycle
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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.
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