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研究生:詹繕合
研究生(外文):Shan-Ho Chan
論文名稱:低劑量X光照射人類肺纖維母細胞所致之旁觀者效應基因表現的研究
論文名稱(外文):Gene Expression Analysis of Low Dose X-ray Induced stander Effects in Human Lung Fibroblasts
指導教授:楊素珍楊素珍引用關係
指導教授(外文):Su-Jing Yang
學位類別:碩士
校院名稱:中臺科技大學
系所名稱:放射科學研究所
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:2007
畢業學年度:95
語文別:中文
論文頁數:79
中文關鍵詞:人類肺纖維母細胞MTT 分析旁觀者效應基因表現分析臨床劑量輻射線
外文關鍵詞:diagnostic radiationsbystander effectsMTT assaygene expression analysishuman lung fibroblasts (MRC-5 and VA-13)
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本研究在評估以臨床劑量輻射線,照射於實驗室培養之人類肺部纖維母細胞株MRC-5、VA-13後,細胞之間產生的旁觀者效應。我們經過數個繼代培養的細胞,讓細胞接受不同劑量的臨床輻射線(0.1Gy及0.25Gy)的照射,照射後再培養24小時後,將取得各個照射過的條件化培養液,以此條件化培養液處理未照射過的細胞,並以MTT技術測量細胞生長存活。我們的發現經臨床劑量輻射線照射纖維母細胞,不論是正常(未轉型) MRC-5或是轉型VA-13的細胞,都會產生20%~40%的生存曲線下降的狀況,因此,纖維母細胞經輻射線照射後,會有旁觀者效應。以接受0.25 Gy的劑量所得條件化培養液處理的72小時細胞,有比較明顯的旁觀者效應。在此條件下,抽取對照組(control) 及實驗組(bystander effect)細胞的total RNA,將兩組RNA作基因微陣列實驗,分析其中表現過度(up-regulation)和基因表現減少(down-regulation)的表現異常基因。這些表現異常的基因,造成細胞的變化,正常(未轉型) MRC-5有14.2%的基因表現變異,轉型的VA-13細胞有11.6%的基因表現變異,不論細胞型態為何,都會造成細胞產生基因的變化,我們也比較了這些基因表現的變異在正常與轉型細胞的異同。
Bystander effect is a sort of indirect biological effect induced by low-dose radiation. However, the molecular mechanism underlies this effect is not fully understood. In this study, we use low-dose radiation of diagnostic source to determine whether it can cause bystander effect on two cell lines MRC-5 and VA-13, which are normal and transformed human lung fibroblasts, respectively. We obtained irradiation-conditioned medium (ICM) by irradiating cells in culture flask and harvesting culture medium 24 h post irradiation and then treated un-irradiated cells with ICM and monitored their survival using MTT assay. We found that both MRC-5 and VA-13 had decrease of 20-30% in cell survival when cells were treated with ICM of 0.25 Gy (~4 abdomen CT dose irradiation) for 72 hour, indicating significant bystander effect in such cultured cells. We further used microarray gene expression profiling assay to analyze the differentially expressed genes in response to such effect. To sum up, normal MRC-5 cells have 14.2% differently expression genes,while transformed VA-13 cells have 11.6%.We further compared patterns of differential expression in respose to bystander medium of above two types of cells, and their related biological meanings are discussed.
中文摘要.........................................................................................I
Abstract.. ...................................................................................... II
致謝……......................................................................................III
內容目錄......................................................................................IV
表目錄….................................................................................... VII
圖目錄…...................................................................................... IX
第一章前言................................................................................ 1
1.1 前言................................ ................................ .......................... 1
1.2 實驗設計................................ ................................ .................. 2
第二章文獻回顧........................................................................ 9
2.1 旁觀者效應................................ ................................ .............. 9
2.2 低劑量輻射造成哺乳動物細胞的旁觀者效應..................... 11
2.3 微陣列分析................................ ................................ ............ 14
2.4 老鼠基因易受輻射致癌的影響確認................................ ..... 16
2.5 生殖細胞突變的敏感性會隨著低劑量輻射的敏感性增加.. 16
2.6 從使用DNA microarray 的基因表現取得基因和路徑劑量反
應圖之方法................................ ................................ ............ 17
2.7 參考文獻所使用的材料與射源................................ ............. 18
第三章材料與方法.................................................................. 20
V
3.1 材料................................ ................................ ........................ 20
3.1.1 材料(一) ................................ ................................ ... 20
3.1.2 材料(二) ................................ ................................ ... 20
3.2 實驗方法:細胞活性測試分析(MTT assay) .................. 22
3.2.1 旁觀者效應實驗流程................................ ................... 23
3.2.2 射源種類與型態................................ ........................... 24
3.3 實驗方法:Target labeling ................................ ................... 26
3.3.1 cDNA 合成與螢光標記................................ ................ 26
3.3.2 基因表現強度訊號的過濾................................ ............ 27
第四章研究結果...................................................................... 29
4.1 結果(一):不同細胞型態的生存曲線................................ 29
4.2 結果(二):細胞型態的改變................................ ............... 31
4.3 結果(三):旁觀者效應的基因微陣列表現分析................ 34
4.3.1 肺部纖維母細胞之旁觀者效應相關表現分析............. 35
4.3.2 MRC-5、VA-13 人類肺部纖維母細胞的表現異常重覆基
因................................ ................................ ................... 43
4.3.3 表現異常趨勢在MRC-5 和VA-13 相反的基因(DEG)44
4.4 結論................................ ................................ ........................ 46
第五章研究討論與建議.......................................................... 47
5.1 討論................................ ................................ ........................ 47
VI
5.1.1 討論(一)細胞照射時吸收劑量的準確度................ 48
5.1.2 討論(二)射源照射與模擬假體的設計.................... 51
5.1.3 討論(三)細胞對輻射的反應................................ .... 52
5.1.4 討論(四)表現基因功能分析................................ .... 53
5.2 建議................................ ................................ ........................ 59
5.2.1 其他可考慮的射源................................ ....................... 59
5.3 未來的驗證方法................................ ................................ .... 60
參考文獻...................................................................................... 62
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