基于γH2AX的免疫荧光分析技术定量检测CT照射后人外周血单个核细胞的DNA损伤
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摘要
第一部分:γH2AX分析定量检测CT照射后人外周血单个核细胞DNA损伤的体外实验研究
目的:以γH2AX的荧光显像和定量检测为依据,通过体外实验研究,初步了解CT辐射剂量与人外周血单个核细胞(PBMCs)γH2AX焦点数量之间的相关性。
材料和方法:67名志愿者纳入研究,签署知情同意书后,分别经肘静脉采集外周血标本,分装至抗凝管中,在不同的剂量下进行体外照射,照射后5分钟对血标本进行一系列实验室处理:分离单个核细胞,固定,破膜,封闭,染色,测定γH2AX的表达。荧光显微镜下观察计数γH2AX焦点,分析其与CT辐射剂量之间的关系。CT辐射剂量参数使用DLP值(剂量长度乘积)。统计学分析方法采用秩和检验、t检验、线性相关和回归分析。
结果:体外CT照射后5分钟人外周血PBMCsγH2AX焦点数量较基底水平(空白对照)显著增加(剂量阈值>199.64 mGy?cm),通过荧光显微镜检测到的γH2AX焦点数量与DLP值呈线性正相关,直线回归方程模型为:Y=0.1917X+0.1248,有显著统计学意义(P=0.000 R2=0.906)。
结论:体外经一定剂量CT照射后,人外周血PBMCs DNA损伤诱发H2AX活化产生γH2AX,其数量与CT辐射剂量有较高的相关性。
第二部分:体内外经CT照射后外周血单个核细胞γH2AX焦点数量的初步临床研究
目的:通过对临床患者CT照射后外周血单个核细胞(PBMCs)γH2AX焦点数量进行体内外实验研究,对比分析γH2AX焦点数量的差异及可能产生机制,引进一种新的生物剂量标记物——γH2AX,进一步探讨γH2AX分析的作为生物剂量计应用前景。
材料和方法:选取符合标准的15名临床CT检查患者为研究对象。签署知情同意书后,CT检查前经肘静脉采集血样4ml/人,按2ml/管分装入2个抗凝管中,标记为①空白对照组,②体外受照组。第①管血样置于37℃孵育箱中以保持细胞活性,备处理。第②管血样固定于病人体表的扫描范围中心,使其始终处于照射野内,扫描结束后取出第②管血样,置入37℃孵育箱中保持细胞活性,备处理;CT检查完成后5分钟,经患者肘静脉再行采集血样2ml/人,编号为③体内受照组,立即同步对3组血样进行一系列实验室处理:分离单个核细胞,固定,破膜,封闭,染色,测定γH2AX的表达。荧光显微镜下观察计数γH2AX焦点,进行对比分析。统计学方法采用t检验、线性相关和回归分析。
结果:体内、外受照组CT照射后5分钟的外周血PBMCsγH2AX焦点数量均与DLP值呈线性正相关,相同DLP值时体外受照组的γH2AX焦点数量明显高于体内组(P=0.000<0.05)。
结论:γH2AX分析在检测CT辐射剂量方面具有独特优势,是一种新型的生物剂量标记物,其作为生物剂量计在对低剂量电离辐射的研究中有广阔的应用前景。
Part one: An experimental study in vitro onγH2AX focus assay to quantify the DNA damage induced in peripheral blood mononuclear cells after CT
Purpose: To prospectively determine ifγH2AX (phosphorylated form of H2AX histone variant)-based visualization and quantification of DNA damage induced in peripheral blood mononuclear cells (PBMCs) can be used to estimate the radiation dose received after computed tomography by in vitro study, comprehend the correlation between the dose and the inducedγH2AX foci initially.
Materials and Methods: Sixty-seven people were recruited prospectively; After written informed patient consent were obtained, blood samples were taken from the antecubital vein of all volunteers, and collected into vacuum tubes containing lithium heparin. The blood samples (in tubes) from each volunteer were exposed to different radiation doses with GE LightSpeed VCT scanner in vitro. 5 minutes later, blood samples were isolated, fixed, and stained forγH2AX expression. TheγH2AX focus yields were determined with fluorescence microscopy, and the radiation doses delivered during CT as dose-length product––DLP. Data were analyzed by using Kruskal–Wallis test, independent–samples t test, linear correlation and regression method.
Results: We found a significantly higher number ofγH2AX foci than control check in blood samples 5 minutes after the last exposure to ionizing radiation at CT(dose threshold exceeds 199.64 mGy?cm), and the focus increase after CT depended linearly on the DLP. The regression equation model is: Y=0.1917X+0.1248, there were statistical difference(P=0.000 R2=0.906).
Conclusion: The DNA damage of PBMCs after CT in vitro is always followed by the formation ofγH2AX, its yields and CT radiation doses are thought to be high correlates.
Part two: Preliminary clinical study of theγH2AX foci levels after in vivo and in vitro CT irradiation in peripheral blood mononuclear cells
Purpose: To determine whetherγH2AX yield analysis can be used as a sensitive biologic dosimeter of human exposure to low-level radiation through comparing H2AX phosphorylation in the blood cells between in vivo and in vitro, and explore its prospect.
Materials and Methods: Fifteen patients scheduled to undergo diagnostic CT were recruited and prospectively entered into the study. After written informed patient consent were obtained, blood samples (4ml per patient) were taken from the antecubital vein of all patients before they underwent CT, and collected into vacuum tubes containing lithium heparin(2ml per tube). The blood samples split into two groups: control check (ck) and in vitro. The ck group was incubated at 37°C. While the in vitro group was fixed on patient’s body surface of central exposure field during the CT scanning, then taken out and incubated at 37°C. 5 minutes later, another blood samples (2ml per patient) were taken from the antecubital vein of all patients named as in vivo group. All of the blood samples were isolated, fixed, and stained forγH2AX expression. TheγH2AX focus yields were determined with fluorescence microscopy, making a comparative analysis. Data were analyzed by using paired–samples t test, linear correlation and regression method.
Results: TheγH2AX focus yields increase 5 minutes after the last exposure to ionizing radiation at CT depended linearly on the DLP in both in vivo and in vitro groups. Furthermore, the PBMCs exposed in vitro represent a higher yields ofγH2AX focus than in vitro on the same condition of DLP(P=0.000<0.05).
Conclusion:γH2AX focus assay’s peculiarly sensitivity and its demonstrated applicability to CT examinations allow the investigation of people who are exposed to well defined radiation doses. It is a novel quantitative biomarker. Thus, the assessment ofγH2AX foci can serve as a biologically relevant biomarker for exposure and provides an exciting perspective to low levels of ionising radiation.
目的:以γH2AX的荧光显像和定量检测为依据,通过体外实验研究,初步了解CT辐射剂量与人外周血单个核细胞(PBMCs)γH2AX焦点数量之间的相关性。
材料和方法:67名志愿者纳入研究,签署知情同意书后,分别经肘静脉采集外周血标本,分装至抗凝管中,在不同的剂量下进行体外照射,照射后5分钟对血标本进行一系列实验室处理:分离单个核细胞,固定,破膜,封闭,染色,测定γH2AX的表达。荧光显微镜下观察计数γH2AX焦点,分析其与CT辐射剂量之间的关系。CT辐射剂量参数使用DLP值(剂量长度乘积)。统计学分析方法采用秩和检验、t检验、线性相关和回归分析。
结果:体外CT照射后5分钟人外周血PBMCsγH2AX焦点数量较基底水平(空白对照)显著增加(剂量阈值>199.64 mGy?cm),通过荧光显微镜检测到的γH2AX焦点数量与DLP值呈线性正相关,直线回归方程模型为:Y=0.1917X+0.1248,有显著统计学意义(P=0.000 R2=0.906)。
结论:体外经一定剂量CT照射后,人外周血PBMCs DNA损伤诱发H2AX活化产生γH2AX,其数量与CT辐射剂量有较高的相关性。
第二部分:体内外经CT照射后外周血单个核细胞γH2AX焦点数量的初步临床研究
目的:通过对临床患者CT照射后外周血单个核细胞(PBMCs)γH2AX焦点数量进行体内外实验研究,对比分析γH2AX焦点数量的差异及可能产生机制,引进一种新的生物剂量标记物——γH2AX,进一步探讨γH2AX分析的作为生物剂量计应用前景。
材料和方法:选取符合标准的15名临床CT检查患者为研究对象。签署知情同意书后,CT检查前经肘静脉采集血样4ml/人,按2ml/管分装入2个抗凝管中,标记为①空白对照组,②体外受照组。第①管血样置于37℃孵育箱中以保持细胞活性,备处理。第②管血样固定于病人体表的扫描范围中心,使其始终处于照射野内,扫描结束后取出第②管血样,置入37℃孵育箱中保持细胞活性,备处理;CT检查完成后5分钟,经患者肘静脉再行采集血样2ml/人,编号为③体内受照组,立即同步对3组血样进行一系列实验室处理:分离单个核细胞,固定,破膜,封闭,染色,测定γH2AX的表达。荧光显微镜下观察计数γH2AX焦点,进行对比分析。统计学方法采用t检验、线性相关和回归分析。
结果:体内、外受照组CT照射后5分钟的外周血PBMCsγH2AX焦点数量均与DLP值呈线性正相关,相同DLP值时体外受照组的γH2AX焦点数量明显高于体内组(P=0.000<0.05)。
结论:γH2AX分析在检测CT辐射剂量方面具有独特优势,是一种新型的生物剂量标记物,其作为生物剂量计在对低剂量电离辐射的研究中有广阔的应用前景。
Part one: An experimental study in vitro onγH2AX focus assay to quantify the DNA damage induced in peripheral blood mononuclear cells after CT
Purpose: To prospectively determine ifγH2AX (phosphorylated form of H2AX histone variant)-based visualization and quantification of DNA damage induced in peripheral blood mononuclear cells (PBMCs) can be used to estimate the radiation dose received after computed tomography by in vitro study, comprehend the correlation between the dose and the inducedγH2AX foci initially.
Materials and Methods: Sixty-seven people were recruited prospectively; After written informed patient consent were obtained, blood samples were taken from the antecubital vein of all volunteers, and collected into vacuum tubes containing lithium heparin. The blood samples (in tubes) from each volunteer were exposed to different radiation doses with GE LightSpeed VCT scanner in vitro. 5 minutes later, blood samples were isolated, fixed, and stained forγH2AX expression. TheγH2AX focus yields were determined with fluorescence microscopy, and the radiation doses delivered during CT as dose-length product––DLP. Data were analyzed by using Kruskal–Wallis test, independent–samples t test, linear correlation and regression method.
Results: We found a significantly higher number ofγH2AX foci than control check in blood samples 5 minutes after the last exposure to ionizing radiation at CT(dose threshold exceeds 199.64 mGy?cm), and the focus increase after CT depended linearly on the DLP. The regression equation model is: Y=0.1917X+0.1248, there were statistical difference(P=0.000 R2=0.906).
Conclusion: The DNA damage of PBMCs after CT in vitro is always followed by the formation ofγH2AX, its yields and CT radiation doses are thought to be high correlates.
Part two: Preliminary clinical study of theγH2AX foci levels after in vivo and in vitro CT irradiation in peripheral blood mononuclear cells
Purpose: To determine whetherγH2AX yield analysis can be used as a sensitive biologic dosimeter of human exposure to low-level radiation through comparing H2AX phosphorylation in the blood cells between in vivo and in vitro, and explore its prospect.
Materials and Methods: Fifteen patients scheduled to undergo diagnostic CT were recruited and prospectively entered into the study. After written informed patient consent were obtained, blood samples (4ml per patient) were taken from the antecubital vein of all patients before they underwent CT, and collected into vacuum tubes containing lithium heparin(2ml per tube). The blood samples split into two groups: control check (ck) and in vitro. The ck group was incubated at 37°C. While the in vitro group was fixed on patient’s body surface of central exposure field during the CT scanning, then taken out and incubated at 37°C. 5 minutes later, another blood samples (2ml per patient) were taken from the antecubital vein of all patients named as in vivo group. All of the blood samples were isolated, fixed, and stained forγH2AX expression. TheγH2AX focus yields were determined with fluorescence microscopy, making a comparative analysis. Data were analyzed by using paired–samples t test, linear correlation and regression method.
Results: TheγH2AX focus yields increase 5 minutes after the last exposure to ionizing radiation at CT depended linearly on the DLP in both in vivo and in vitro groups. Furthermore, the PBMCs exposed in vitro represent a higher yields ofγH2AX focus than in vitro on the same condition of DLP(P=0.000<0.05).
Conclusion:γH2AX focus assay’s peculiarly sensitivity and its demonstrated applicability to CT examinations allow the investigation of people who are exposed to well defined radiation doses. It is a novel quantitative biomarker. Thus, the assessment ofγH2AX foci can serve as a biologically relevant biomarker for exposure and provides an exciting perspective to low levels of ionising radiation.
引文
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6. Rothkamm K, Balroop S, Shekhdar J, et al. Leukocyte DNA Damage after Multi- Detector Row CT: A Quantitative Biomarker of Low-Level Radiation Exposure. Radiology 2007; 242(1):244-251.
7. Geisel D, Heverhagen JT, Kalinowski M, et al. DNA Double-Strand Breaks after Percutaneous Transluminal Angioplasty. Radiology 2008; 248(3):852-859.
8. Fernandez Capetillo O, Lee A, Nussenzweig M, et al. H2AX: the histone guardian of the genome. DNA Repair 2004; 3(8-9):959-967.
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1. United Nations Scientific Committee on the Effects of Atomic Radiation. Medical radiation exposures: UNSCEAR 2000 report to the General Assembly. New York, NY: United Nations, 2000:295-495.
2. Brenner DJ, Hall EJ. Computed tomography—an increasing source of radiation exposure. N Engl J Med, 2007, 357:2277-2284.
3.刘必跃.试述CT剂量的安全及管理.计量技术,2007,5:58-60.
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2. Brenner DJ, Hall EJ. Computed tomography—an increasing source of radiation exposure. N Engl J Med, 2007, 357:2277-2284.
3.刘必跃.试述CT剂量的安全及管理.计量技术,2007,5:58-60.
4. McNitt-Gray MF. AAPM/RSNA physics tutorial for residents - topics in CT: radiation dose in CT. Radiographics, 2002, 22:1541-1553.
5. Huda W. Radiation doses and risks in chest computed tomography examinations. Proc Am Thorac Soc, 2007, 4:316-320.
6. Hart D, Wall BF. UK population dose from medical x-ray examinations. Eur J Radiol, 2004, 50:285-291.
7. Louis C.德国人正在为CT的过度使用而担心.放射学实践, 2007, 22:829.
8. Dixon AK,Goldstone KE.Abdominal CT and the euratom directive. Eur Radiol, 2002, 12:1567-1570.
9. International Commission on Radiological Protection. Managing patient dose in Multi-Detector Computed Tomography (MDCT). Publication 102, Annals of the ICRP, 2007, 37.
10. Huda W, Vance A. Patient radiation doses from adult and pediatric CT. Am J Roentgenol, 2007, 188:540-546.
11. Virginia T, John E, Raju S, et al. Dose reduction in CT while maintaining diagnostic confidence: diagnostic reference levels at routine head, chest, and abdominal CT-IAEA-coordinated research project. Radiology, 2006, 240:828-834.
12. Shrimpton PC, Hillier MC, Lewis MA, et al. National survey of doses from CT in the UK: 2003. Br J Radiol, 2006, 79:968-980.
13. Health risks from exposure to low levels of ionizing radiation—BEIRⅦ. Washington, DC: National Academies Press, 2005.
14. Brooks AL. From cell to organism: the need for multiparametric assessment of exposure and biological effects. Br J Radiol, 2005, 27:S139-145.
15. Rothkamm K, Balroop S, Shekhdar J, et al. Leukocyte DNA damage after multi–detector row CT: a quantitative biomarker of low-level radiation exposure. Radiology, 2007, 242:244-251.
16. Yoshinaga S,Mabuchi K,Sigurdson AJ,et al. Cancer risks among radiologists and radiologic technologists: review of epidemiologic studies. Radiology, 2004, 233:313- 321.
17. Cardis E, Vrijheid M, Blettner M, et al. The 15-country collaborative study of cancer risk among radiation workers in the nuclear industry: estimates of radiation-related cancer risks. Radiat Res, 2007, 167:396-416.
18. Berrington de Gonzalez A, Darby S. Risk of cancer from diagnostic X-rays: estimates for the UK and 14 other countries. Lancet, 2004, 363:345-351.
19. Preston DL, Pierce DA, Shimizu Y, et al. Effect of recent changes in atomic bomb survivor dosimetry on cancer mortality risk estimates. Radiat Res, 2004, 162:377-389.
20. Preston DL, Ron E, Tokuoka S, et al. Solid cancer incidence in atomic bomb survivors: 1958-1998. Radiat Res, 2007, 168:1-64.
21. Brenner DJ,Elliston CD. Estimated radiation risks potentially associated with full-body CT screening. Radiology, 2004, 232:735-738.
22. Brenner DJ. Radiation risks potentially associated with low-dose CT screening of adult smokers for lung cancer. Radiology, 2004, 231:440-445.
23. Brenner DJ, Elliston CD, Hall E, et al. Estimated risks of radiation-induced fatal cancer from pediatric CT. AJR, 2001, 176:289-296.
24. Giles J. Study warns of‘avoidable’risks of CT scans. Nature, 2004, 431:391.