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研究生:蕭雅燕
研究生(外文):Ya-Yan Hsiao
論文名稱:以微陣列技術探討p53基因突變之腦瘤細胞的輻射效應
論文名稱(外文):The Effects of Radiation on p53-Mutated Glioma Cells Using cDNA Microarray Technique
指導教授:吳國海
指導教授(外文):Frank Q.H. 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:2003
畢業學年度:91
語文別:英文
論文頁數:65
中文關鍵詞:微陣列分析p53細胞週期停滯輻射線效應反轉錄聚合脢鏈鎖反應
外文關鍵詞:Microarrayp53cell cycle arrestradiation effectReverse Transcription-Polymerase Chain Reaction
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本研究主要是探討放射線引發腦腫瘤細胞的放射生物效應,我們選用的細胞為一p53基因突變的惡性腦瘤細胞株(U138MG),照射計量為10格雷,並且在照射後不同的時間點(範圍包含30分鐘到48小時)收取細胞作細胞週期的分析,我們利用流式細胞分析儀以及紅色螢光物質PI (propidium iodide) 偵測DNA的含量;借由細胞群落形成與否來推定細胞的存活及增殖能力;同時用雙色螢光微陣列技術進一步探究細胞被放射線引發的基因分子機制,在細胞照射後的5個時間點(30分鐘、6、12、24、36小時)觀察基因表現的變化,其中微陣列實驗採用了安捷倫公司的基因晶片,該產品包含了12814個人類基因片段。細胞週期的分析結果顯示放射線照射後,會使該細胞週期逐漸停滯於G2/M期,而凋亡的細胞隨時間的延續有漸漸變多趨勢。在照射後36小時,在重組細胞群中,細胞週期的停滯現象會逐漸消退,此時,部分(10%)的細胞走向死亡衰退途徑,其餘的則是存活下來並且進行細胞週期的運轉。由微陣列分析的結果,我們對於放射線引起細胞的放射生物效應機制作了以下的推定:該現象是由一群細胞週期調控基因(像是p21及cyclin B1等基因)與細胞死亡相關基因(如tumor necrosis factors與BBC3等基因)作用的結果。同時,放射線引發U138MG細胞的G2/M細胞週期停滯現象的訊息傳遞是經由p21與GADD45A但不必依靠p53基因調控。本研究亦發現當p53功能喪失時,U138MG細胞會利用其他p53家族成員(像是TP63、TP73)來調節p21等下游基因而將細胞週期停滯在G2/M期。然而,仍有一些分子機制層面的訊息傳遞基因尚待進一步分析;在未來,可再運用西方墨點法與免疫沉澱分析等蛋白質研究技術,將基因表現層次與蛋白質層次作連結。
In this study, we investigated the effects of 10-Gy -irradiation on cell-cycle arrest, apoptosis and clonogenic death in the p53-mutated human U138MG (malignant glioblastoma) cell line. In order to evaluate time-dependent events in cellular responses to radiation, we did a time course study by incubating cells ranging from 0.5 to 48 hours after irradiation. Cell-cycle distribution and apoptosis were evaluated by flow cytometry using propidium iodide (PI) staining. Cell viability and proliferative capacity were studied by colony formation assay. Dual fluorescence cDNA microarray technique was used to examine the differential expression patterns of the irradiated cells at five time points (0.5, 6, 12, 24, and 36 h) post-irradiation. The cDNA microarray chips (Agilent Human I_clonesetB2) used contained DNA sequences corresponding to 12,814 human genes. From the flow cytometry data, we observed that after 10-Gy irradiation, cell-cycle checkpoint for U138MG cells were predominantly at G2/M phase, reached a maximum of about 86% at 36 h and there was no appearent arrest at G1/S or S phase. Late apoptosis was more evident after irradiated cells released from G2/M arrest. Microarray analyses revealed changes in the expression of a number of cell-cycle-related genes (p21, cyclin B1, etc.) and cell-death genes (tumor necrosis factors, BBC3, etc.) suggesting their involvement in radiation-induced cell-cycle arrest and apoptosis. Validation of the microarray results was made using semiquantitative RT-PCR technique. Based on our transcription results and related established knowledge, we have proposed a p53-independent pathway model to explain the extensive G2/M arrest observed in irradiated U138MG cells.
Chinese Abstract.………………...….………………………………....1
English Abstract.…………………….….……………………………...2
Abbreviations……..........................……………………………………3
I. Introduction....…………………..…..……………………………4
II. Materials and Methods.………..…………………………………7
1. Cell culture
2. Growth Curve
3. Survival Curve
4. DNA Sequencing of p53
5. Cell Cycle Distribution Analysis
6. cDNA Microarray Expression Analysis
7. Data Analysis
8. Semiquantitative Reverse Transcription Polymerase Chain Reaction
III. Results………………….…….…………………………………..23
IV. Disscussion …………….….……………………………………..27
V. Conclusions………..……………………………………………..33
VI. References………………………………………………………..34
VII. Tables..……….…………………………………………………..42
VIII. Figures………..…………………………………………………..45
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