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研究生:蘇聖芳
研究生(外文):Sheng-Fang Su
論文名稱:高低劑量率之低劑量游離輻射導致hprt基因突變頻率及分佈之探討
論文名稱(外文):Evaluation of hprt Mutant Frequency and Mutation Spectrum Induced by High and Low Dose-rate Low Doses of Ionizing Radiation
指導教授:黃正仲黃正仲引用關係
指導教授(外文):Jeng-Jong Hwang
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:2004
畢業學年度:92
語文別:中文
中文關鍵詞:低劑量輻射低劑量率輻射hprt基因突變頻率缺失分析
外文關鍵詞:Low Doses RadiationLow Dose-rate Radiationhprt geneMutant FrequencyDeletion Assay
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過去大部分對於低劑量游離輻射所可能造成的生物效應之研究,往往是藉由高劑量游離輻射對細胞存活分率的結果再反推回低劑量區而得到的資訊。因此, 所得到的低劑量生物效應並非完全正確且貼近於在一般環境中低劑量且低劑量率的暴露。為了瞭解低劑量率低劑量的輻射暴露所引起的基因突變是否與高劑量率低劑量有所差異, 本研究主要探討中國倉鼠卵巣10B2細胞株 (CHO-10B2) 在高低劑量率低劑量游離輻射照射後,次黃嘌呤磷酸核醣基轉化酶 (hypoxanthine-gaunine phosphoribosyl transferase, hprt) 基因之突變頻率及分布。
以高低劑量率Co-60射源分別將細胞照射至0, 0.2, 0.5, 0.75 及1.0 Gy, 並以6-硫氫基鳥糞嘌呤 (6-thioguanine, 6-TG) 來篩選hprt基因功能缺失的突變株 (hprt-)。由於hprt基因有九個表現子 (exons),並已有研究指出游離輻射大都會造成基因大片段的缺失, 因此我們進一步利用複合聚合酶鏈鎖反應 (multiplex polymerase chain reaction, M-PCR) 來分析hprt基因突變的情形。並且針對hprt九個exon皆未發現缺失之突變基因進行DNA定序 (sequencing) 分析。另外, 更以細胞週期分析及處於分裂期細胞之比例來觀察高低劑量率低劑量游離輻射照射後細胞分布的情形。
本研究結果顯示, 無論在高或低劑量率, 低劑量的游離輻射照射下, hprt基因突變頻率皆隨劑量增加而增加, 並在累積劑量為0.2 Gy時, 發現有高輻射敏感反應 (hyper-radiosensitivity) 及反劑量率效應 (inverse dose-rate effect) 的存在。相對於自然誘發hprt基因之突變大多屬於點突變, 輻射誘發之突變大多數為缺失突變, 其中又以末端缺失為多。同時在0.2 Gy時, 缺失突變之突變頻率也發現較0.5 Gy為高。另外, 定序結果顯示大多數的突變在hprt exon 3-9的位置, 以 A及T的改變為多, 且有22%為啞突變 (silent mutation)。
根據我們的認知, 本實驗結果首次發現, 0.2 Gy低劑量照射下的中國倉鼠卵巣細胞有異常升高的hprt基因突變頻率, 明顯不同於”接受的劑量愈低則突變頻率愈少”的傳統觀念。同時以hprt突變頻率來量測細胞接受1 Gy以下的生物效應, 在過去的文獻中也沒有被報告過。因此經由hprt基因突變分布的分析, 期望應可用來評估人類細胞在高低劑量率低劑量游離輻射曝露下, 基因傷害的形式及分子機制, 並有助於鑑定基因傷害的程度。
Radiobiological effect under low doses of most studies have been obtained by back extrapolating the cell survival from high doses. It does not demonstrate an accurate response of the effects of most environmental exposures, which tend to be of low dose and protracted over time.
For this reason, the effect of high and low dose-rate (HDR, LDR), low doses ionizing radiation (IR) on the mutant frequency (MF) and the mutation spectrum at the hypoxanthine phosphoribosyl transferase (hprt) locus was studied in Chinese hamster ovary (CHO-10B2) cell line. Cells were cultured in the medium containing 6-thioguanine (6-TG) for 14 days to select for hprt mutants following 0, 0.2, 0.5, 0.75 and 1.0 Gy Co-60 γ- ray exposure. The mutation spectrum was determined for exon deletion analysis by multiplex polymerase chain reaction (M-PCR). Intact mutants were further analyzed by sequencing. In addition, cell cycle distribution analysis and mitotic cells were confirmed by flow cytometry and mitotic index (MI), respectively.
The results showed that hprt MF was increased with radiation doses under both HDR and LDR irradiation. However, the higher MF induced by 0.2 Gy suggests that hyper-radiosensitivity (HRS) and inverse dose-rate effect likely exist around this dose range. The dose response for the induction of hprt large deletions were significantly increased with radiation dose (r2=0.99, p=0.0064) compared to the point mutation (r2=0.29, p=0.35) induced by LDR IR which is the major of spontaneous mutations. In addition, exons deletions were found mostly at both ends of hprt gene. Terminal deletions were the predominant mutation type (r2=0.95, p=0.02). The higher deletion mutation at 0.2 Gy under LDR irradiation was also found. Furthermore, base sequencing of intact mutations seemed to indicate that hprt exons 3-9, and bases A or T were mostly substituted and about 22% were silent mutations.
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