以核磁共振技術的代謝體學研究游離輻射對人類淋巴細胞之影響

1H NMR-based Metabolomics to Study Effects of Ionizing Radiation in Human Lymphocytes

劉姮君(Heng-Chun Liu)

指導教授 ： 林靖愉

共同指導教授 ： 蔡孟勳

國立臺灣大學/公共衛生學院/環境衛生研究所/碩士(2012年)

https://doi.org/10.6342/NTU.2012.02005

摘要

雖然流行病學調查已證實暴露游離輻射會對人體有不良效應，也已證實會有引發癌症的風險。然而，即使在醫療上利用游離輻射作為診斷與治療的工具已有相當多發展，對於游離輻射的生物效應的機制目前仍有許多不了解之處。為了瞭解游離輻射對生物系統的影響，近年來已有許多利用功能性基因體學的方法研究游離輻射對生物體內小分子層面的效應。而利用代謝體學方法探討游離輻射影響生物系統中代謝物變化的研究並不多，因此，本研究希望藉由利用代謝體學的方法瞭解游離輻射對於代謝體效應。本研究利用兩種不同p53狀態的人類淋巴細胞：TK6及WTK1（TK6細胞的p53為正常狀態，WTK1則是突變型的p53）來進行游離輻射的代謝物效應研究。將細胞分別暴露不同劑量的γ射線：TK6及WTK1都暴露 D0劑量（TK6為0.8 Gy，WTK1為1.5 Gy）、及WTK1細胞暴露10 Gy，並於暴露後收集經過3小時或24小時的細胞樣本以進行劑量效應及時間效應的探討。細胞萃取後分為水溶性及脂溶性代謝物兩層，都以核磁共振儀進行1H及J-resolved實驗分析。並將數據結果利用主成分分析、單因子變異數分析及Student’s t-tes找出可能統計上重要的代謝物並進行代謝物鑑定。本研究的結果發現，TK6及WTK1兩細胞株的水溶性代謝物及脂溶性代謝物受游離輻射的影響有明顯不同。而在兩細胞株以及不同劑量下都可以發現，水溶性代謝物的時間效應中，經過24小時的變化比經過3小時的變化更顯著。游離輻射對WTK1造成的劑量效應可能與細胞內氧化壓力有關。而研究結果中發現游離輻射影響代謝物變化可能與細胞內的氧化壓力、細胞膜受損、細胞凋亡以及DNA受損有關。牛磺酸、丙胺酸、肌酸及榖胱甘肽在TK6及WTK1間的變異可能來自於兩細胞p53狀態的不同。

關鍵字

核磁共振儀 ； 人類淋巴細胞 ； 游離輻射 ； p53 ； 代謝體學

並列摘要

Although epidemiological studies have demonstrated adverse human health effects with a greater risk for cancer development when subjects are exposed to ionizing radiation, the mechanisms of biological impacts are still unclear despite the increasing use of diagnostic radiology in medicine. In recent years, several functional genomic approaches have been developed and applied to examine molecular events in biological systems exposed to ionizing radiation. Little metabolomic studies were conducted on the metabolic effects of ionizing radiation. Therefore, we intend to use metabolomic approach to understand the molecular events in a more functional measurement. Human lymphoblastic cell lines: TK6 (wild-type p53) and WTK1 (mutant p53) were used to examine metabolic effects of ionizing radiation. Time-course and dose-response experiments were conducted in cells treated with gamma-ray （iso-survival dose, D0 (TK6: 0.8 Gy; WTK1: 1.5 Gy） or 10 Gy) for 3 or 24 hours. Hydrophilic and hydrophobic metabolites were extracted and analyzed by 1H and J-resolved NMR followed by principal component analysis and metabolite identification. Based on our results, both hydrophilic and hydrophobic metabolome are shown different radiation effects between TK6 and WTK1 cells. The γ-ray radiation effects on hydrophilic metabolome at 24 hours are greater than at 3 hours both in different cell types and doses. The dose effect of ionizing radiation on the WTK1 may be related to intracellular oxidative stress. In summary, after γ-ray exposure, the hydrophilic metabolome changing in cells may caused by oxidative stress, cell membrane damage, apoptosis and DNA damage. The various p53 status also results in differences in taurine, alanine, creatine and glutathione between TK6 and WTK1 cells.

並列關鍵字

nuclear magnetic resonance (NMR) ； human lymphocytes ； ionizing radiation ； p53 ； metabolomics

參考文獻

Abe, M., M. Takahashi, K. Takeuchi, and M. Fukuka. 1968. Studies on the significance of taurine in radiation injury. Radiation research. 33:563-573.

Albers, M.J., R. Bok, A.P. Chen, C.H. Cunningham, M.L. Zierhut, V.Y. Zhang, S.J. Kohler, J. Tropp, R.E. Hurd, Y.F. Yen, S.J. Nelson, D.B. Vigneron, and J. Kurhanewicz. 2008. Hyperpolarized 13C lactate, pyruvate, and alanine: noninvasive biomarkers for prostate cancer detection and grading. Cancer research. 68:8607-8615.

Amores-Sanchez, M.I., and M.A. Medina. 1999. Glutamine, as a precursor of glutathione, and oxidative stress. Molecular genetics and metabolism. 67:100-105.

Anthony, M.L., M. Zhao, and K.M. Brindle. 1999. Inhibition of phosphatidylcholine biosynthesis following induction of apoptosis in HL-60 cells. The Journal of biological chemistry. 274:19686-19692.

Beckonert, O., H.C. Keun, T.M.D. Ebbels, J. Bundy, E. Holmes, J.C. Lindon, and J.K. Nicholson. 2007. Metabolic profiling, metabolomic and metabonomic procedures for NMR spectroscopy of urine, plasma, serum and tissue extracts. Nature Protocols 2:2692 - 2703.