臺灣博碩士論文加值系統

對於輻射潛在致死損傷(radiation potential lethal damage, PLD)之分子機制，三十年來一直不甚清楚。本研究試圖應用高密度寡核酸微陣列技術及生物資訊學來解答輻射照射後0.5M NaCl 高張溶液處理對於人類腦神經膠質瘤U87MG 細胞株之PLD 之影響。微陣列實驗結果顯示，於20,173 個基因中，有210 個基因之表現量明顯改變，根據生物功能分類顯示其與代謝及能量、訊息傳遞、運輸、局部化、刺激反應、細胞內反應過程調控、細胞型態發生、免疫反應、細胞週期、神經生理過程、細胞生長、細胞死亡等功能有關。其中，有一組位於粒線體DNA 上之基因表現被抑制，包括MTND1、MTND3、MTCO1、MTCO2 和MTATP8 這5 個基因之共同抑制表現會導致粒線體之ATP 合成功能失常，經由細胞實驗也確認細胞內粒線體膜電位的確有降低的情形發生。同時，粒線體轉譯機轉之抑制及ATP 能量耗損本身會全面影響細胞正常運作並引發細胞壞死(necrosis)。此外，我們也確認部分細胞會經由衰老(senescence)的方式走向死亡。此一能量缺損病變，應可解釋固定PLD 會增進細胞死亡之原因，同時也說明為何在高張溶液處理後DNA 修補蛋白運輸會被抑制。此外，H2AX 磷酸化之測定則發現DNA 之雙股螺旋斷裂顯著增加，此一觀察與細胞生存曲線中α 值增加相符。DNA content 分析結果則顯示細胞週期檢查點改變(cell-cycle checkpoints) 與高張溶液增進細胞死亡亦相關。本研究結果將有助於解答固定輻射潛在致死損傷的分子機制以及細胞死亡方式，同時這也是利用系統性生物學高通量微陣列技術得到的新發現。

The molecular mechanism underlying radiation potentially lethal damage (PLD) has been of great interest, yet remains unclear, in radiation biology for more than 30 years. In this study, we applied high-density oligonucleotide microarray and bioinformatics to investigate the hypertonic effect on PLD in U87MG glioma cells. Analysis of the microarray data showed that the expression levels of 210 genes out of 20,173 genes were altered. Classification of these affected 210 genes according to their biological functions indicated that they are involved in metabolism/energy, cell communication/signaling, transport, localization, response to stimuli, cell organization biogenesis, morphogenesis, immune response, cell cycle, neurophysiological process, cell proliferation and cell death. In addition, a unique set of mitochondrial genes responsible for ATP production including MTND1, MTND3, MTCO1, MTCO2 and MTATP8 were down-regulated. Inhibition of mitochondria transcription machinery and ATP exhaustion will affect cell's normal physiology and cause necrosis. Cell death through senescence was also observed. Such catastrophe action due to energy depletion following hypertonic treatment may explain not only the enhanced cell killing, but the inhibited repair protein mobility as well. Phosphorylated H2AX assay was also related with the increase of DNA double strand breaks (DSBs), which was consistent with the increased α component in the survival response curve. DNA histogram showed that the enhanced cell killing may depend on cell-cycle checkpoints. This investigation with systemic biology and high-throughput microarray may be helpful in elucidating molecular mechanism underlying PLD fixation.

壹. 中文摘要………………………………………………….- 4 -
貳. ABSTRACT………………………………………………- 5 -
叁. 前言……………………………………………………….- 6 -
潛在致死傷害( potentially lethal damage, PLD)與高張溶液環境………………………...- 6 -
細胞對輻射照射的反應與histone H2AX 的磷酸化…………………………...………...- 7 -
腦神經膠質瘤(Glioblastoma Multiforme)………………………………………….……....- 8 -
細胞死亡……………………………………………………………………………...….….- 9 -
肆. 材料與方法……………………………………………….- 10 -
U87MG 細胞株………………………………………………………………………...…..- 10 -
潛在致死傷害固定現象(PLD Fixation)…………………………………………...………- 11 -
細胞存活曲線……………………………………………………………………......…….- 12 -
細胞週期分析………………………………………………………………………...……- 12 -
測量DNA 損傷………………………………………………………………………….…- 13 -
粒線體基因表現壓抑現象的測量…………………………………..……………..…...…- 13 -
測量細胞內的粒線體內膜間電位改變……………………………………...………...….- 16 -
細胞死亡相關實驗…………………………………………………………………...……- 16 -
以不同的分析方法重新分析microarray data………………………………...……..……- 20 -
實驗數據的統計分析………………………………………………………………...……- 21 -
伍. 實驗結果與討論…………………………………………..- 22 -
U87MG 細胞株生長狀態………………………………………………...………………- 22 -
輻射照射後 高張/等張 溶液處理對細胞存活曲線的影響………………………….….- 22 -
透過流體細胞儀對細胞的細胞週期分布進行分析……………………………….……..- 24 -
透過流體細胞儀對細胞中γH2AX 的含量進行分析………………………….…………- 25 -
透過流體細胞儀對活細胞內粒線體內膜間電位(Mitochondrial Proton potential)高低進行分
析…………………….…………………………………………………………………..…- 27 -
利用即時定量PCR 驗證Microarray Data( Real-Time PCR )……………………...…….- 30 -
利用Caspase 3 Activity Assay 對細胞是否有經由apoptosis 走向死亡進行分析……...- 31 -
利用Annexin V / PI 雙染Assay 來測量不同時間點中有多少比率的Apoptosis 細胞…- 32 -
利用TUNEL Assay 測量有多少百分比細胞是以Apoptosis 走向死亡………………..…- 35 -
利用Trypan Blue Exclusion Assay 測量不同時間點necrosis 細胞佔所有細胞中百分比- 38 -
利用SA β-Gal Assay 測量細胞經由Senescence 走向死亡的比率…………………….….- 40 -
運用不同分析技巧重新分析欣茹的Microarray Data………………………………….…..- 42 -
陆. 結論………………………………………………………..- 51 -
柒. 參考文獻:………………………………………………….- 53 -
捌. Figures................................................................................ - 61 -
玖. Tables...................................................................................- 82 -
Appendix_A- Figure (Fit KEGG pathway of 210 genes)………...….- 86 -
Appendix_A- Table (Functional Group analysis of 210 genes)….…..- 95 -
Appendix_B (Hsin-Ju thesis data).......................................................- 112 -
Appendix_C (Mammalian Cell Death)................................................- 116 -

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