BrdU法研究放射性核素内照射诱发HPRT基因突变

目的：研究放射性核素内照射诱发大鼠外周血淋巴细胞HPRT基因突变的剂量效应关系。方法：大鼠尾静脉注入晚期混合裂变产物，5－溴脱氧尿嘧啶（BrdU）法检测不同累积剂量和不同剂量率内照射诱发外周血淋巴细胞HPRT基因突变，并拟合剂量效应关系。结果：随着累积剂量和剂量率的增加，HPRT基因突变频率不断上升，其剂量效应关系符合线性模型，结论：BrdU法是一种快速，简便，较敏感的检测辐射诱发HPRT基因突变的方法，HPRT基因突变可以作为辐射生物剂量计。     

放射性核素内照射诱发大鼠外周血淋巴细胞HPRT基因位点突变与染色体畸变的研究

目的 阐明放射性核素内照射诱发外周血淋巴细胞HPRT基因位点突变的剂量效应关系, 并与染色体畸变剂量效应关系进行比较。方法 给动物尾静脉注射放射性核素, 注射量为0.5ml/100g体重。剂量效应关系组动物注射活度为3.64×10⁵Bq/ml, 于注射后1、3、6和9d心脏穿刺取血。剂量率效应关系组动物注射活度分别为3.64×10⁵、1.82×10⁵、0.91×10⁵和0.445×10⁵Bq/ml, 于注射后3、6.7、17和42d心脏穿刺取血。应用多核细胞法及胞质分裂阻断法(CBMN)和常规染色体畸变分析法检测HPRT基因位点突变率和染色体畸变率。用计算机拟合剂量效应关系和剂量率效应关系函数。结果 淋巴细胞HPRT基因位点突变率不仅随内照射剂量和剂量率增加而增加, 呈现出良好的正相关, 而且与染色体畸变亦呈现出较好的相关性。结论 辐射诱发HPRT基因位点突变有可能成为有效的辐射生物剂量计。     

晚期混合裂变产物内照射诱发大鼠外周血淋巴细胞HPRT基因位点突变与微核的比较研究

目的　探索ＨＰＲＴ 基因位点突变检测作为辐射生物剂量计的可行性。方法　运用多核细胞法及胞质分裂阻断微核（ＣＢＭＮ） 法研究大鼠外周血淋巴细胞的ＨＰＲＴ基因位点突变及微核与累积剂量、剂量率之间关系。结果　静注晚期混合裂变产物后第１ 天（ 累积剂量１－７３ ｃＳｖ） ,与对照组相比,ＨＰＲＴ 位点变异频率（Ｖｆ）、微核细胞（ ＭＮＣ） 率、微核（ ＭＮ） 率均已显著增加（Ｐ＜ ０－０１）。ＨＰＲＴＶｆ、ＭＮＣ率、ＭＮ 率与累积剂量间均可拟合成对数回归方程：Ｙ＝ ａ＋ ｂｌｎＸ。在总累积剂量近似相等条件下,ＨＰＲＴ Ｖｆ、ＭＮ率与剂量率之间的关系均可用函数Ｙ＝ ａ ＋ ｂｌｎＸ 表示。ＨＰＲＴ Ｖｆ 与ＭＮ 率间存在线性相关。结论　ＨＰＲＴ基因位点对晚期混合裂变产物内照射非常敏感,有明显的剂量效应关系。ＨＰＲＴ 基因突变检测可望成为评估电离辐射损伤的生物剂量计。     

裂片核素内照射诱导大鼠外周血淋巴细胞DNA单链断裂

目的：探讨混合裂变产物致癌的分子机理和混合裂变产物内污染诱发免疫细胞的放射免疫毒理效应。方法：应用碱性单细胞凝胶电泳技术观察了混合裂变产物急性染毒时对大鼠外周血淋巴细胞DNA的损伤。结果：裂片核素内照射对大鼠外周血淋巴细胞DNA具有明显的损伤作用，慧星细胞率和DNA的损伤程度随染毒时间的延长、累积剂量增大而逐渐加重，且存在较好的剂量效应关系。结论：单细胞凝胶电泳技术检测的DNA单链断裂可作为毒物对健康影响的早期生物学指标。     

X射线诱发外周血淋巴细胞TCR基因突变研究

目的 用培养法研究X射线诱发的人外周血淋巴细胞TCR 基因突变情况。方法 以不同剂量(0~8 Gy) 的X射线照射新鲜分离的健康成人外周血淋巴细胞, 植物血凝素、白细胞介素2(IL-2)协同刺激培养7 d, 流式细胞术检测TCR基因突变频率(TCR MF),并拟合剂量效应关系。结果 随着照射剂量的增加,TCR基因突变频率随之上升, 最佳拟合曲线为二次多项式模型。结论 TCR 基因突变可作为辐射生物剂量计,用于急性辐射照射生物剂量的估算。     

放疗前后鼻咽癌患者淋巴细胞损伤的研究

目的研究放射治疗对鼻咽癌患者外周血淋巴细胞的损伤效应，探讨染色体畸变率和HPRT基因位点突变成为辐射生物剂量计的可行性。方法对鼻咽癌患者放疗前后外周血淋巴细胞染色体畸变率、HPRT基因位点突变频率分别进行检测分析。结果鼻咽癌患者放疗前外周血淋巴细胞染色体畸变率、HPRT基因位点突变频率与对照组及放疗后相比差异均有显著统计学意义。结论外周血淋巴细胞染色体畸变率、HPRT基因突变频率联合进行检测可望成为评价放疗致机体遗传学损伤的辐射生物剂量计。     

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。     

不同剂量率X射线辐照对小鼠免疫系统的影响

目的 检测经相同剂量不同剂量率X射线照射的BalB／C小鼠外周血淋巴细胞周期及胸腺和脾脏指数。方法 18只BalB／c小鼠随机分为对照组（controI），低剂量率照射组（20cGy／min）和高剂量率照射组（300cGy／min），每组6只。低剂量组和高剂量组采用剂量率分别为2．0cGy／min和300cGy／min的1GyX射线对小鼠进行全身照射，24h后取血及器官，用流式细胞仪检测外周血淋巴细胞的周期变化，用称量的方法得到胸腺和脾脏指数。结果 高剂量率辐射时，小鼠外周血淋巴细胞的损伤较低剂量时大，而且对雄性鼠的影响大于雌性；同时，胸腺和脾脏指数变化也随着剂量率的增大而减小。结论 低剂量率的照射对小鼠外周血淋巴细胞、胸腺和脾脏的影响较高剂量率辐射小；雌性鼠的辐射耐受能力较雄性强。     

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。     

辐射致大鼠平滑肌细胞HPRT基因第7/8外显子突变分析

目的探讨辐射剂量与平滑肌细胞HPRT基因变异的关系,提供放射预防血管再狭窄的基础实验依据.方法不同剂量的放射性核素188Re内辐射平滑肌细胞后,应用6-TG筛选分离HPRT变异细胞,采用PCR-SSCP技术进行HPRT基因7/8外显子的变异分析.结果辐射后平滑肌细胞HPRT基因突变率为(5.5～13)×10-6;91株HPRT突变细胞克隆中,14.3%呈现7/8外显子缺失,16.5%呈现点突变,7/8外显子总变异率为30.8%;辐射剂量与HPRT基因突变率、第7/8外显子的总变异率及外显子缺失率呈正相关.结论辐射所致DNA分子的损伤,包括基因片段的缺失、断裂及点突变与辐射剂量呈正相关.     

Low dose X-radiation adaptive response in spleen and prostate of Atm knockout heterozygous mice

PURPOSE: To investigate the effect of being heterozygous for a knockout mutation in the ataxia telangiectasia (Atm) gene on radiation adaptive response. MATERIALS AND METHODS: DNA recombination, as measured by pKZ1 inversion frequency, was quantified by histochemistry in Atm knockout heterozygous prostate and spleen 3 days after treatment with a priming dose of 0.01 or 10 mGy X-radiation 4 h prior to a challenge dose of 1,000 mGy. RESULTS: In spleen and prostate, a single dose of 0.01 mGy caused an induction in inversion frequency but a dose of 10 mGy prevented the induction of a proportion of endogenous inversions. Both doses induced an adaptive response, of similar magnitude, to a subsequent high challenge dose for chromosomal inversions in both spleen and prostate. The adaptive response completely prevented the induction of inversions from a 1,000 mGy challenge dose and also a proportion of endogenous inversions. The adaptive responses and distribution of inversions across gland cross-sections observed here in Atm knockout heterozygote prostate were similar to those induced in Atm wild-type prostate in a previous study. CONCLUSIONS: Being heterozygous for a knockout mutation in the Atm gene does not affect the endogenous pKZ1 inversion frequency, the inversion response to single low radiation doses used here, or the induction of a radiation adaptive response for inversions in pKZ1 mouse spleen or prostate.     

Radiation effects in lymphocytes of children living in a Chernobyl contaminated region of Belarus

Purpose : To investigate cytogenetic and mutational eÚects in lymphocytes from individuals chronically exposed to radiation from the Chernobyl catastrophe. Materials and methods : Nine years after the Chernobyl accident (1986), peripheral blood lymphocytes from 20 Kalinkovichi children (age 10-15) and 10 Minsk children (age 10-17) were analysed for genetic damage by several assays. Radiation damage in exposed children was investigated in descendants of progenitor cells that were irradiated during a short period immediately after the accident. In the time-span between the accident and blood sampling the cells were also irradiated chronically by internal radiation originating from ingested radionuclides and, to a smaller extent, by external radiation from radionuclides. The parameters measured in whole blood smears were the frequency of micronucleated mononucleated lymphocytes and binucleated lymphocytes with nucleoplasmic bridges and associated micronuclei. Cultures of cytokinesis-blocked lymphocytes were used to analyse mononuclear and binuclear cells for the presence of micronuclei, also cell killing effects. A colony assay was used to study induction of recessive mutations in the HPRT gene. Results : The analysis of whole-blood smears indicated a doubling of the frequency of micronuclei per 100 mononuclear lymphocytes in exposed children compared with unirradiated children. Small numbers of binucleated lymphocytes with nucleoplasmic bridges and associated micronuclei were found in blood smears from exposed children. Analysis of cytokinesis-blocked cultures indicated in mononuclear cells of exposed children a statistically significant increase in the frequency of micronuclei. When the same parameters were studied in binucleated cells there was no difference between exposed and unexposed children. Results of the dye-exclusion assay showed a four-fold increase in the percentage of dead cells between exposed and unexposed children. There was no evidence for induction of HPRT mutations in exposed children. Conclusion : These results indicate that the frequently advocated procedure of simply analysing micronuclei in cytokinesis-blocked binucleated lymphocytes can result in an underestimate of genetic damage induced by radiation accidents. Biodosimetric studies should therefore employ a battery of assays for the detection of several types of genetic damage in different generations of lymphocytes.     

Radiation effects in lymphocytes of children living in a Chernobyl contaminated region of Belarus

PURPOSE: To investigate cytogenetic and mutational effects in lymphocytes from individuals chronically exposed to radiation from the Chernobyl catastrophe. MATERIALS AND METHODS: Nine years after the Chernobyl accident (1986), peripheral blood lymphocytes from 20 Kalinkovichi children (age 10-15) and 10 Minsk children (age 10-17) were analysed for genetic damage by several assays. Radiation damage in exposed children was investigated in descendants of progenitor cells that were irradiated during a short period immediately after the accident. In the time-span between the accident and blood sampling the cells were also irradiated chronically by internal radiation originating from ingested radionuclides and, to a smaller extent, by external radiation from radionuclides. The parameters measured in whole blood smears were the frequency of micronucleated mononucleated lymphocytes and binucleated lymphocytes with nucleoplasmic bridges and associated micronuclei. Cultures of cytokinesis-blocked lymphocytes were used to analyse mononuclear and binuclear cells for the presence of micronuclei, also cell killing effects. A colony assay was used to study induction of recessive mutations in the HPRT gene. RESULTS: The analysis of whole-blood smears indicated a doubling of the frequency of micronuclei per 100 mononuclear lymphocytes in exposed children compared with unirradiated children. Small numbers of binucleated lymphocytes with nucleoplasmic bridges and associated micronuclei were found in blood smears from exposed children. Analysis of cytokinesis-blocked cultures indicated in mononuclear cells of exposed children a statistically significant increase in the frequency of micronuclei. When the same parameters were studied in binucleated cells there was no difference between exposed and unexposed children. Results of the dye-exclusion assay showed a four-fold increase in the percentage of dead cells between exposed and unexposed children. There was no evidence for induction of HPRT mutations in exposed children. CONCLUSION: These results indicate that the frequently advocated procedure of simply analysing micronuclei in cytokinesis-blocked binucleated lymphocytes can result in an underestimate of genetic damage induced by radiation accidents. Biodosimetric studies should therefore employ a battery of assays for the detection of several types of genetic damage in different generations of lymphocytes.     

Low dose X-radiation adaptive response in spleen and prostate of Atm knockout heterozygous mice

Purpose: To investigate the effect of being heterozygous for a knockout mutation in the ataxia telangiectasia (Atm) gene on radiation adaptive response.

Materials and methods: DNA recombination, as measured by pKZ1 inversion frequency, was quantified by histochemistry in Atm knockout heterozygous prostate and spleen 3 days after treatment with a priming dose of 0.01 or 10 mGy X-radiation 4 h prior to a challenge dose of 1000 mGy.

Results: In spleen and prostate, a single dose of 0.01 mGy caused an induction in inversion frequency but a dose of 10 mGy prevented the induction of a proportion of endogenous inversions. Both doses induced an adaptive response, of similar magnitude, to a subsequent high challenge dose for chromosomal inversions in both spleen and prostate. The adaptive response completely prevented the induction of inversions from a 1000 mGy challenge dose and also a proportion of endogenous inversions. The adaptive responses and distribution of inversions across gland cross-sections observed here in Atm knockout heterozygote prostate were similar to those induced in Atm wild-type prostate in a previous study.

Conclusions: Being heterozygous for a knockout mutation in the Atm gene does not affect the endogenous pKZ1 inversion frequency, the inversion response to single low radiation doses used here, or the induction of a radiation adaptive response for inversions in pKZ1 mouse spleen or prostate.