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研究生:黃佰璋
研究生(外文):Po-Chang Huang
論文名稱:游離輻射及無機砷誘發細胞凋亡之研究
論文名稱(外文):Apoptosis induced by ionizing radiation and inorganic arsenic
指導教授:郭浩然郭浩然引用關係王應然王應然引用關係
指導教授(外文):How-Ran GuoYing-Jan Wang
學位類別:碩士
校院名稱:國立成功大學
系所名稱:環境醫學研究所
學門:醫藥衛生學門
學類:公共衛生學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:51
中文關鍵詞:細胞週期細胞凋亡X射線微矩陣晶片三氧化二砷放射線
外文關鍵詞:chemotherapyAs2O3radiationX-rayprogrammed cell deathcell cyclebiochipmicroarrayradiationapoptosisU937
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輻射及無機砷是存在於我們生活環境中有礙健康的物質。游離輻射所產生的能量可以打斷DNA的雙股螺旋,進而造成遺傳物質的變異,甚至造成生物體立即性的傷害。此外,長期暴露在三氧化二砷的環境中,也已被證實為導致烏腳病及其他癌症的危險因子。即便如此,適量的X射線以及三氧化二砷仍可作為治療癌症之用。X射線照射可導致癌細胞的凋亡;具有相同作用的三氧化二砷,也已被用來治療白血病病人。但是,接受放射線治療的病人會產生發燒、嘔吐、食慾不佳等症狀;而長期服用三氧化二砷的病人,也會併發一些臨床症狀,包括有黃胆症狀的肝臟傷害,及肝細胞癌的發生等。

本研究之目的為利用不同劑量的X射線或三氧化二砷,及結合低劑量的三氧化二砷與X射線,處理人類單核球(monocyte)細胞株 U937更有效率的細胞凋亡。以期作為未來治療癌症之參考。

我們以trypan blue對放射線及三氧化二砷處理過的細胞染色,觀察細胞的生長率的變化,再以電泳分析DNA碎裂片段片段碎裂的情形,及利用flow cytometry對DNA碎裂片段做定量分析。同時,我們也以cDNA微矩陣晶片來探討,經15Gy放射線照射之U937細胞,其細胞凋亡過程中基因之表現。

本研究首先發現U937 細胞與放射線照射的劑量反應關係。在高劑量(15 Gy) 照射後6-8 小時,細胞開始凋亡;在低劑量(4 Gy) 照射,細胞約於36-48 小時開始凋亡。以基因晶片分析細胞接受15 Gy 照射後12 小時,初步發現有791 個基因被過度表現,但有183 個基因被抑制;細胞接受15 Gy 照射後4 小時,發現有408個基因被過度表現,3 個基因被抑制。此外,我們再以Clontech corp.的生物晶片產品,以相同的劑量及時間點做分析。

在低劑量三氧化二砷處理過的U937,可觀察到細胞生長率高於對照組;而以2GyX射線照射過後,再以低劑量三氧化二砷處理的U937細胞,則觀察到細胞生長有被抑制的趨勢。Caspase 3, glutathione S-transferase pi, SAFB, 和 Topisomerase-I以晶片分析的結果是有過度表現,而HSP60, Cyclin B1, 和 NFKB2則是被抑制。未來這些被過度表現或抑制的基因,可用來進一步作為癌症治療的標的物。本研究的限制為使用生物晶片需要高額的經費,但使用晶片能在短時間內大量篩檢有興趣的基因。
Ionizing radiation and inorganic arsenic are both human health hazards existing in the natural environmental. The high energy emitted by ionizing radiation can break the double helix of DNA and thus induce the gene mutation, which may cause the instantaneous biological damage. Chronic exposure to arsenic trioxide (As2O3) had been proven to be a risk factor of blackfoot disease and cancers. On the other hand, usage of proper dosage of As2O3 and X-ray may help curing cancers. The principle of radiotherapy of cancer is to kill the tumor cells by causing apoptosis. Likewise, As2O3 can be applied to treat acute promyelocytic leukemia through the apoptosis pathway. However, patients treated with radiation therapy may develop symptoms including vomiting, fever, and poor appetite, and patients ingesting arsenical medicines may also develop symptoms such as degenerative lesions with jaundice and even hepatocellular carcinoma.

To further understand effects of ionizing radiation and arsenic on apoptosis, we applied combinations of various dosages of X-ray and arsenic trioxide to treat U937 cells. We hope our results may provide more information in setting the proper strategy of cancer treatment in the future.

To assess the survival of cells, U937 cells were stained with trypan blue, and their number was determined by microscopic calculation. In addition, DNA fragmentation was demonstrated by gel electrophoresis. Flow cytometry technique was used to quantify the apoptosis cells in the population. cDNA microarray was used to analyze the expression of apoptosis related genes after cells were exposed to 15 Gy of X-ray.

A dose-response relationship was discovered between X-ray exposure and apoptosis of U937 cells. After 6-hours incubation, DNA fragmentation was detected in U937 cells exposed to 15 Gy X-ray; however, DNA fragmentation in cells exposed to 4 Gy X-ray could be found only after 36-hours incubation. The results were consistent with our flow cytometry data. From the results of temporal gene expression of U937 cells exposed to 15 Gy of X-ray, 408 genes were found to be over-expressed and 3 genes were down regulated at 4 hours, and 791 genes were over-expressed and 183 genes were down-regulated at 12 hours.

The cell viability is better in the U937 cells treated with less than 1 uM of As2O3 in comparison with those received dosages higher than 1 uM. When treated with both X-ray and As2O3, the cell growth rate was lower than the control. Caspase 3, glutathione S-transferase pi, SAFB, and Topisomerase-I were found to be over expressed in our data analysis, but HSP60, Cyclin B1, and NFKB2 were down regulated. By reviewing the relevant articles, our data can completely explain the situation of apoptosis. Some genes were supposed to be candidates of biomarkers for cancer therapy in the future. The limitation of using cDNA microarry is its high cost, but its strength is to screen a large number of genes in a relatively short period of time. Few, if any, studies using both X-ray and As2O3 to induce and explore the tumor cell lines’ apoptosis have been conducted before. Our study identified changes in the expressions of a number of apoptosis related genes through the application of these two inducers in conjuction with cDNA microarry, which is a powerful tool to screen a large number of genes at the same time.
圖目錄 v
表目錄 vi
研究摘要
中文摘要 1
英文摘要 2
誌謝 4
第一章 研究介紹
第一節 背景資料 5
第二節 研究目的 8
第三節 研究意義及重要性 8
第二章 研究材料及方法 9
第三章 研究結果
第一節 X射線照射U937 17
第二節 生物晶片(BioChip)篩選細胞凋亡相關基因 18
第三節 以三氧化二砷及X射線處理U937細胞 19
第四章 討論 20
第五章 參考文獻 24
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