山东济宁辐射事故受照人员生物剂量估算及彗星电泳检测分析

目的探索特大剂量照射后外周血和骨髓染色体培养方法，拟合6Gy以上大剂量照射染色体双着丝点＋环剂量-效应曲线，对山东济宁“10．21”事故受照者进行准确生物剂量估算和DNA损伤检测。方法采集2例受照者外周血和骨髓细胞，制备染色体标本，计数双（多）着丝点＋环数目；用正常离体人血拟合6～22Gy双＋环剂量效应曲线及数学方程；对2例事故受照者进行生物剂量估算。用碱性单细胞凝胶电泳方法检测受照者外周血DNA损伤。结果B的外周血染色体双＋环平均数为4．47个／细胞；A的外周血培养无分裂细胞，骨髓染色体双＋环平均数为9．15个／细胞。用6—22Gy剂量效应方程估算全身平均受照剂量，B为9．4Gy，A为19．5Gy。单细胞凝胶电泳可见2例受照者的多数彗星细胞呈小头大尾形状。结论用新建立的6～22Gy染色体畸变剂量效应曲线估算2例受照者的生物剂量，已分别达到极重度骨髓型放射病和肠型放射病水平。     

Okadaic acid诱导早熟凝集染色体测定辐射生物剂量

目的建立人外周血的早熟凝集染色体（prematurely condensed chromosomes，PCC）环与辐射剂量之间的剂量．效应关系曲线。方法取健康成年人外周血，^60Coγ射线分别以0、1、2、5、10、15、20和25Gy照射，吸收剂量率为1Gy／min，培养48h，终止培养前1h加入Okadaic acid诱导早熟凝集染色体，观察人外周血淋巴细胞PCC环与剂量效应的关系。结果Okadaic acid诱导的人外周血淋巴细胞早熟凝集染色体环随着辐射剂量的增加而增加，但在20Gy以后达到饱和。结论在20Gy剂量范围内，PCC环与辐射剂量具有良好的剂量．效应关系，与双着丝粒相比，它可作为大剂量受照情况下的生物剂量指示剂。     

河南“4．26”^60Co源辐射事故受照者的生物剂量（染色体畸变）估算

目的：用染色体畸变分析方法对河南“4．26”^60Co辐射事故7例受照者，进行了早期生物剂量估算，方法：照后4－5d取血培养，分析第一次有丝分裂细胞“双＋环”畸变率，并由此估算生物剂量，用“双＋环”畸变在细胞间的泊松分布情况，检验照射的均匀性，结果：7例受照者依据“双＋环”畸变率估算的个体辐射剂量分别为5．09Gy(梅），2.61Gy（天），2.49Gy(旺），0.89Gy/(勇），0.70Cy(民），0.58Gy（义）和0.08Gy(宇），与用物理方法测定的剂量化比较接近，亦与放射损伤的临床诊断完全吻合，泊松分布检验证实，“梅”和“旺”双＋环畸变离泊松分布，基余5例符合泊松分布，结论：染色体畸变分析是非常可靠的生物剂量估算方法。“梅”和“旺”受到不均照射，基他5例受到比较均匀的照射。     

电离辐射诱导端粒延长作用的研究

目的观察人细胞系A549照射后端粒酶活性的变化及其对端粒长度的影响。方法采用端粒重复序列扩增法（TRAP）检测照射前、后不同时间点细胞端粒酶活性，端粒限制性片段平均长度分析法（耵谭）检测端粒长度。结果在1—5Gy剂量范围内A549端粒酶活性表达呈剂量和时间依赖性增强；照射后TRF延长，照射剂量越高，TRF延长出现越早，随着时间的延长，较低剂量组耵谭也明显延长，表明这种诱导作用与端粒酶表达增强有相近似的剂量效应和时间效应。结论端粒放射损伤可通过端粒序列的合成（延长端粒）进行修复；端粒酶可能在端粒放射损伤修复中起到重要作用。     

3例60Co源辐射事故病人受照生物剂量(微核法)估算

目的：用胞质分裂阻滞微核法（CBMN）对河南“4.26”^60Co源辐射事故中3例受照者进行生物剂量估算,方法：照后10d取血培养,0.3ml全血加入到4ml培养基中,37℃培养44h加松胞素B素终常浓度为6mg.L^-1,继续教育28h收获制片,CBMN以千分数表示,结果：根据照后10dCBMN频率估算受照者“梅”,“天”和“旺”的生物学剂量分别为5．45Gy,2.84Gy t 2.78Gy,该剂量与染色体双＋环及物理剂量相近,与临床放射损伤的诊断也相符合,结论：在放射事故中检测CBMN可作为生物剂量计。     

60Coγ射线超大剂量照射离体人外周血染色体畸变的剂量-效应研究

目的 探讨超大剂量γ射线离体照射后人外周血淋巴细胞染色体畸变的量效关系,建立双+环(dic+r)大剂量-效应曲线。方法 外周静脉血样采自3名健康男性,经 ⁶⁰Co γ线(0~50 Gy,剂量率2.35Gy/min)照射。采用即刻加秋水仙素的微量全血法,分别培养52、72及96h。计数有丝分裂指数(MI)、双着丝粒体(dic)和环(r)畸变数,拟合dic+r剂量-效应曲线,对2例受大剂量照射的事故患者进行剂量估算。结果 MI随照射剂量的增加而逐渐减少。每细胞dic+r频率随照射剂量增加而增加直到23Gy(5Gy之后增加幅度较前变小),＞23Gy后趋于饱和。对所获数据进行回归分析,拟合dic+r大剂量-效应曲线(5~23Gy):每细胞dic+r频率y=-1.608(±0.300)+0.830 (±0.051)D-0.013(±0.002) D² (R²=0.998)。用拟合曲线对2例受照患者剂量的估算结果与用物理方法和电子自旋共振(ESR)法估算的剂量及临床表现基本一致。结论 本研究建立的dic+r大剂量-效应曲线, 可估计的上限剂量达23Gy,有可能提高常规染色体畸变分析用作生物剂量计的实用价值。     

γ射线和裂变中子诱发用或不用PHA刺激人淋巴细胞的微核

在有丝分裂期间不能掺入子核的染色体或染色体断片,以微核形式存留在胞质中.辐射能诱发微核,并证明离体照射血细胞,微核与剂量存在着良好的剂量-效应关系.采用Fenech和M?rley提出的用松胞素B(Cyt-B)阻断胞质分裂的方法,研究正常人淋巴细胞在低剂量率(LDR)和高剂量率(HDR)γ射线以及低剂量率的~(252)Cf照射后所诱发的经PHA(植物血凝素)刺激和不经PHA刺激淋巴细胞的微核率.     

不同剂量率γ射线照射诱发淋巴细胞早熟凝集染色体环产额的研究

目的 比较不同剂量率γ射线照射诱发的淋巴细胞早熟凝集染色体环(PCC-R)的产额,建立不同剂量率的人外周血早熟凝集染色体环与辐射剂量之间的剂量效应曲线。方法 取健康成年人外周血,分别用吸收剂量率为0.5和1.0Gy/min的 ⁶⁰Co γ 射线照射,吸收剂量为 0、 1、 2、 5、 10、 15、 20和25Gy。培养48h,终止培养前1h加入冈田酸(okadaic acid)诱导早熟凝集染色体,观察人外周血淋巴细胞PCC-R产额与照射剂量的关系。结果 在20 Gy剂量范围内,人外周血淋巴细胞PCC-R产额随着剂量的增加而增加。在25 Gy剂量范围内,在相同剂量情况下,吸收剂量率为1.0 Gy/min的PCC-R产额都要高于0.5 Gy/min的产额,且在20和25 Gy剂量点的差异有统计学意义。结论 PCC-R作为大剂量受照情况下的生物剂量指示剂,基于不同剂量率建立的剂量效应关系曲线所估算剂量的结果会有所不同。     

3例~(60)Co源辐射事故病人受照生物剂量(微核法)估算

目的　用胞质分裂阻滞微核法 (CBMN)对河南“4 2 6”6 0 Co源辐射事故中 3例受照者进行生物剂量估算。方法　照后 10d取血培养 ,0 3ml全血加入到 4ml培养基中 ,37℃培养 44h加松胞素B最终浓度为 6mg·L- 1 ,继续培养 2 8h收获制片 ,CBMN以千分数表示。结果　根据照后 10dCBMN频率估算受照者“梅”、“天”和“旺”的生物学剂量分别为 5 45Gy、2 84Gy和 2 78Gy。该剂量与染色体双 环及物理剂量相近 ,与临床放射损伤的诊断也相符合。结论　在放射事故中检测CBMN可作为生物剂量计。     

重离子12 C诱发人淋巴细胞染色体畸变和微核的研究

目的 探讨不同LET重离子12C诱发人淋巴细胞的生物效应.方法 采集2名健康男性自愿献血者血样,用60Co γ射线及LET为29和148 keV/μm的12C重离子照射,剂量为0.5～5.0 Gy,剂量率为0.5 Gy/min.采用同时加秋水仙素和松胞素B的方法,检测了淋巴细胞染色体畸变双着丝粒和着丝粒环(双加环)及淋巴细胞微核.结果 29和148 keV/μm的重离子12C诱导染色体畸变和微核的剂量-效应关系分别为线性和线性平方模式.诱发的染色体畸变率随着LET的增高而增高;诱发的微核率剂量＜3.0 Gy,随着LET的增高而增高,剂量＞3.0 Gy微核率趋于饱和.148 keV/μm的12C离子诱导的染色体畸变双加环,随着培养时间的延长,从0.41 ～ 1.32增加到0.68 ～ 3.00.结论 重离子12C诱导的染色体畸变随着时间和LET的增高而增加,微核在一定剂量范围内随着LET的增高而增加,其生物效应比低LET辐射60Co γ射线强.     

优化胞质分裂阻滞实验方案用于核质桥分析的可行性

目的探索优化胞质分裂阻滞实验方案用于辐射诱导核质桥分析的可行性,为以核质桥为指标进行生物剂量估算提供科学依据。方法用2 Gy ⁶⁰Coγ射线(剂量率为1 Gy/min)照射人离体外周血(设0 Gy对照),照射后28 h在细胞样品中加入终浓度为6μg/ml的松胞素B,培养48、56、68和72 h后收获;或照射后加入终浓度为0.6、1、2、6、10μg/ml的松胞素B,培养68 h后收获。应用胞质分裂阻滞法进行标本制备,分析单核细胞、双核细胞、多核细胞的比例,以及辐射诱导核质桥率及微核率。结果对不同细胞培养时间,随细胞培养时间的增加,0和2 Gy核分裂指数和双核细胞比例具有升高的趋势;2 Gy核质桥率无明显变化规律(0.0230~0.0330/细胞),差异无统计学意义(P>0.05),微核率差异亦无统计学意义(P>0.05)。对不同浓度松胞素B处理组,随松胞素B浓度的增加,0和2 Gy时核分裂指数和双核细胞比例有所升高;2 Gy核质桥率无明显变化规律(0.0230~0.0470/细胞),差异无统计学意义(P>0.05);与6μg/ml组相比,10μg/ml组微核率显著降低(U=2.74,P<0.01)。结论不同细胞培养时间组和不同松胞素B浓度组的辐射诱导核质桥率差异无统计学意义。适当缩短细胞培养时间可提前得到核质桥分析结果;培养开始加入松胞素B可简化实验步骤,但可供分析的细胞数过少,用于剂量估算的可行性尚需进一步研究。     

Influence of circadian rhythms on radiosensitivity: single and fractionated dose studies in mouse skin.

In rodent skin significant increases in labelling and mitotic indices have been reported during the period of maximum nocturnal activity. It has been suggested that sparing of radiation damage in fast-proliferating normal tissues could be achieved if treatments were given at the time of day when the maximum number of normal cells were most radioresistant. If changes in radiosensitivity do occur in tissues with circadian fluctuations in the cell kinetic parameters, then the magnitude of these changes should be dependent on the size of dose per fraction. Because of the implications for clinical radiotherapy, especially in regimes where multiple fractions per day are given (MFD), possible diurnal variations in radiosensitivity were investigated using single dose and fractionated X-ray regimens (5F/5 days, 8F/8 days), in rodent skin. Treatments were delivered at 1, 3, 5 a.m. (time of highest DNA synthetic activity in mouse epidermis), 6 and 7 a.m. (highest mitotic activity) and at 5 p.m. (minimum labelling and mitotic indices). To investigate a large range of doses per fraction, fractionated X-rays were given alone or followed by neutron top-up doses. Using a range of doses per fraction of 30 Gy down to 1 Gy, we did not detect any change in radiosensitivity with any of the schedules. Our results suggest that a decrease in normal tissue tolerance is unlikely to be observed in patients even if irradiated at a time of day at which a maximum increase in radiosensitivity might be predicted on the basis of a high mitotic index.     

部分照射离体血对淋巴细胞染色体畸变形成的影响

目的 分析⁶⁰Coγ射线部分照射离体血对人外周血淋巴细胞染色体畸变形成的影响。方法 用2Gy⁶⁰Coγ射线37℃照射人外周血后以不同比例混合后进行培养、制片和分析染色体畸变。结果 染色体畸变随照射血比例的增加而增加,与单纯照射组相比,混合照射血的染色体畸变率高。1:1比例混合估算出的剂量为1.27Gy,大于1Gy;0.5:1比例混合估算出的剂量为0.93Gy,大于0.5Gy;而在1:0组,照射剂量与估算剂量基本一致。结论 染色体畸变可以作为估算非均匀照射的生物学指标之一。     

Simple biodosimetry method for use in cases of high-dose radiation exposure that scores the chromosome number of Giemsa-stained drug-induced prematurely condensed chromosomes (PCC)

There is a need for quick dose estimation by a simple method in radiation accidents. This study develops a simple and rapid dose estimation protocol for victims of such accidents, in particular those involving high radiation doses. Human peripheral blood lymphocytes (PBL) were gamma-irradiated in vitro at several dose points up to 60 Gy, and were stimulated with phytohaemagglutinin-P (PHA-P) for 2 days to obtain dividing cells. PBL were then forced to condense prematurely, using 50 nM calyculin A, and the obtained chromosome spreads were Giemsa stained. The G2-PCC (prematurely condensed chromosomes) index and chromosome number for each radiation dose point were scored. G2-PCC were stably induced using calyculin A within 24 h delays in stimulation of PBL with PHA-P. The chromosome number of G2-PCC increased steeply with radiation doses up to 30 Gy at a rate of 0.31 Gy(-1) and then decreased at 0.30 Gy(-1) up to 40 Gy. More than 10% of G2-PCC index remained up to a 15 Gy dose. Even after 40 Gy irradiation, about 2% PCC index was obtained, and this value was enough to score a sufficient number of chromosome spreads for analysis. Therefore, the combined use of chromosome number and G2-PCC index allows biodosimetry to be done easily and rapidly. If PCC are not induced using calyculin A, it is strongly suggested that the radiation dose is over 50 Gy. A rapid and easy dose estimation for large dose exposure whole-body was realized by combined analysis of Giemsa-stained chromosome number of G2-PCC and PCC index using calyculin A. This simple method will be of use for rapid decision making of therapy for radiation accident victims. This method also has potential for use as a biodosimeter for partial-body exposure accidents.     

不同剂量60Co γ 射线部分照射人离体血对染色体畸变形成的影响

目的 分析不同剂量60Co γ射线部分照射人离体血对淋巴细胞染色体畸变形成的影响.方法 用0～8 Gy(剂量率为0.35 Gy/min)60Coγ射线在37 ℃条件下照射3份离体健康人外周血标本,以0.5∶1的比例与同一供血者的未受照血混合,120 min后进行培养、制片,显微镜下分析染色体畸变(双着丝粒+着丝粒环)的变化,借此进行剂量估算.结果 各组的双着丝粒体+着丝粒环和总畸变,以及断片和单体断裂均随着剂量的增加而增加.用双着丝粒+着丝粒环进行的剂量估算,0.5～2 Gy组大于照射剂量的1/3,4～8 Gy组均接近照射剂量的1/3.结论 染色体畸变可以作为估算非均匀照射的生物学指标之一.

Abstract：

Objective To investigate the effects of 60Co γ-ray partial radiation on chromosome aberration in human peripheral blood in vitro.Methods The samples of heparinized peripheral whole blood from 3 healthy persons were exposed to 60Co γ-rays at the doses between 0 and 8 Gy with the dose rate of 0.35 Gy/min at the temperature of 37 ℃ ,and then mixed with the unirradiated blood samples of the Microscopy was used to observe the chromosome aberration double ( centromere + centromere) and the biological dose was estimated thereby.ResultsThe amounts of double centromere + centromere were increased along with the dose of irradiation in all groups.The estimated biological dose was higher than the 1/3 of the irradiation dose when the dose was between 0.5 to 2 Gy,and was close to the 1/3 of the irradiation dose when the dose was between 4 to 8 Gy.Conclusion Chromosome aberration can be used as a biomarker in estimation of uneven irradiation.     

胞浆分裂阻滞微核法对山东"10·21"辐射事故病人受照射剂量的估算

目的 对山东"10·21"辐射事故中2例严重受照射者进行淋巴细胞微核(MN)检测,并估算受照射剂量.方法 用胞浆分裂阻滞微核(CBMN)法对2例患者(A和B)的外周血和骨髓样本分别进行MN检测.结果 2例患者的外周血培养均未见双核淋巴细胞.患者A的骨髓培养所获双核细胞极少,依据双核淋巴细胞多少粗估剂量＞20Gy.患者B的骨髓MN率为2.42个/细胞,剂量估计为8.7(8.0～9.4)Gy,与用染色体畸变分析、物理方法及ESR法所估算剂量接近,与临床表现基本一致.结论 MN法简便快速,结果准确,是除染色体畸变分析之外又一种可靠的生物剂量计.     

Evaluation of the premature chromosome condensation scoring protocol after proton and X-ray irradiation of human peripheral blood lymphocytes at high doses range

Purpose of the study: One of the main difficulties in radiation dose assessment is cells inability to reach mitosis after exposure to acute radiation. Premature chromosome condensation (PCC) has become an important method used in biological dosimetry in case of exposure to high doses. Various ways to induce PCC including mitotic cells fusion, chemical stimulation with calyculin A or okadaic acid give wide spectrum of application. The main goal of this study was to evaluate the utility of drug-induced PCC scoring procedure by testing 2 experimental modes where 150 and 75 G2/M-PCC phase cells were analyzed after exposure to high dose proton and X-ray radiation. Another aim is to determine the differences in cellular response induced by proton and photon radiation using a HPBL in vitro model as a further extension of our previous studies involving doses up to 4.0?Gy.

Materials and methods: Total body exposure was simulated by irradiating whole blood collected from a healthy donor. Whole blood samples were exposed to two radiation types: 60?MeV protons and 250 kVp X-rays in the dose range of 5.0–20.0?Gy, the dose rate for protons was 0.075 and 0.15?Gy/s for X-rays. Post 48?h of human peripheral blood lymphocytes (HPBL) culture, calyculin A was added. After Giemsa staining, chromosome spreads were photographed and manually analyzed by scorers in the G2/M-PCC phase. In order to check the consistency of obtained results all scorers followed identical scoring criteria. Additionally, PCC index kinetics was evaluated for first 500 cells scored.

Conclusions: Here we provide a different method of results analysis. Presented dose-response curves were obtained by calculating the value of counted excess chromosome fragments. The results indicated that obtained dose estimates as adequate in the high dose range till 18.0?Gy for both studied radiation types, giving an opportunity to further improve PCC assay procedure and shorten the analysis time i.e. in case of partial-body exposure. Moreover, the study presents preliminary results of HPBL cellular response after proton irradiation at high doses range showing differences of PCC index kinetics for different cell classes and cell distribution.     

南京^192Ir放射事故患者照后第4年细胞遗传学随访

目的用5种细胞遗传学方法对南京192Ir源放射事故患者进行照后第4年随访,筛选回顾性剂量估算指标,为探明电离辐射远后效应提供依据。方法采集事故患者外周血,进行非稳定性染色体畸变(双着丝粒+着丝粒环)、微核和核质桥分析,应用荧光原位杂交技术及G显带方法检测染色体易位,并估算生物剂量。结果荧光原位杂交技术及G显带方法检测染色体易位估算的剂量分别为1.45和1.21 Gy,与事故后短期内估算的生物剂量相近;非稳定性染色体畸变、微核、核质桥估算的剂量分别为0.56、0.45、0.41 Gy,低于事故后短期内估算的生物剂量,其修正系数与时间的变化规律符合幂函数模型。结论荧光原位杂交技术及G显带方法检测染色体易位估算方法,适用于回顾性剂量估算,应用非稳定性染色体畸变进行回顾性剂量估算需用修正系数进行校正。     

M-FISH技术检测辐射诱导染色体易位和双着丝粒畸变

目的　探讨用多色荧光原位杂交 (M FISH)技术检测的易位和双着丝粒染色体畸变的差异。方法　用 1,2 ,3,7,8,9,14和 15号染色体端粒和着丝粒特异性探针的M FISH方法 ,分析6 0 Coγ射线离体照射的脐带血淋巴细胞染色体易位和双着丝粒畸变。结果　 (1)用M FISH方法分析6 0 Coγ射线诱导的易位和双着丝粒染色体畸变的剂量 效应曲线 ,均符合线性二次剂量效应模式 ;易位与双着丝粒的比值在大多数剂量水平不等于 1。 (2 )细胞中无非稳定性畸变的完全相互易位的比例随着吸收剂量的增加而降低。 (3)对大多数被标记染色体 ,3 0 0Gyγ射线照射诱发的染色体畸变观察值与理论值无差别 ;9号染色体畸变 (易位和双着丝粒 )观察值显著高于理论值 (P <0 0 5或P <0 0 1) ,15号染色体易位观察值显著低于理论值 (P <0 0 1)。结论　电离辐射诱导的染色体易位率不等于双着丝粒畸变率 ;对于大多数染色体 ,辐射诱发染色体易位率和双着丝粒畸变率符合随机性规律。     

Simple biodosimetry method for use in cases of high-dose radiation exposure that scores the chromosome number of Giemsa-stained drug-induced prematurely condensed chromosomes (PCC)

There is a need for quick dose estimation by a simple method in radiation accidents. This study develops a simple and rapid dose estimation protocol for victims of such accidents, in particular those involving high radiation doses. Human peripheral blood lymphocytes (PBL) were γ-irradiated in vitro at several dose points up to 60?Gy, and were stimulated with phytohaemagglutinin-P (PHA-P) for 2 days to obtain dividing cells. PBL were then forced to condense prematurely, using 50 nM calyculin A, and the obtained chromosome spreads were Giemsa stained. The G2-PCC (prematurely condensed chromosomes) index and chromosome number for each radiation dose point were scored. G2-PCC were stably induced using calyculin A within 24?h delays in stimulation of PBL with PHA-P. The chromosome number of G2-PCC increased steeply with radiation doses up to 30?Gy at a rate of 0.31?Gy^???1 and then decreased at 0.30?Gy^???1 up to 40?Gy. More than 10% of G2-PCC index remained up to a 15?Gy dose. Even after 40?Gy irradiation, about 2% PCC index was obtained, and this value was enough to score a sufficient number of chromosome spreads for analysis. Therefore, the combined use of chromosome number and G2-PCC index allows biodosimetry to be done easily and rapidly. If PCC are not induced using calyculin A, it is strongly suggested that the radiation dose is over 50?Gy. A rapid and easy dose estimation for large dose exposure whole-body was realized by combined analysis of Giemsa-stained chromosome number of G2-PCC and PCC index using calyculin A. This simple method will be of use for rapid decision making of therapy for radiation accident victims. This method also has potential for use as a biodosimeter for partial-body exposure accidents.