染色质结构变化标志物IRIF作为高LETα粒子内照射生物剂量估计指标的研究

The health effects of internal incorporation of alpha radionuclides following human exposure in nuclear accident, nuclear wars and nuclear terrorism and from environmental radon exposure have become a world-widely concerned issue. However, the internally incorporated radionuclides constitutes a particular type of protracted irradiation and their distribution in the body is usually very uneven, which makes the estimate of biological dose very difficult and does not be solved so far in the worldwide. In recent years, convincing evidence indicates that high linear energy transfer (LET) ionizing radiation (IR) induces clustered DNA lesions, a unique class of DNA lesion and double-strand breaks (DSB) clusters increase with LET. For DNA damage, cells have evolved efficient and rapid repair responses to maintain the integrity of the genome. Sensor proteins have been shown to localize to the sites of DSB within seconds to minutes following IR exposure, and then recruit transducer proteins which provide the signals to enzymes to repair the break, resulting in the formation of microscopically visible nuclear domains named the ionizing radiation induced foci (IRIF). Evidence from recent years suggests that chromatin organization mediates the response to DNA damage and persistent IRIF may reveal an aberrant chromatin structure due to illegitimate rejoining. It is noted that the persistent IRIF is heritable to a large progeny of cells leading to the emergence of new and stable phenotypes. In this study, a dose-response curve will be established for persistent IRIF yield as a marker of permanent chromatin alteration resulting from DSB-clustered DNA lesions using immunofluorescence analysis and flow cytometry in human peripheral blood lymphocytes irradiated in vitro with Am-241 source and Rn-222 and its progeny. Whether the persistent IRIF is specific to high-LET α-particle and the differential biological effectiveness among α-particles with different energy will be investigated. The dose-response curve of persistent IRIF yield in mouse peripheral blood lymphocytes and bone marrow cells exposed to Rn-222 and its progeny will be established in vitro respectively, which may estimate the modified factor of in vivo dose between mouse's circulating lymphocytes and red bone marrow cells. The red bone marrow absorbed dose of uranium miners exposed to radon will be estimated by detecting the persistent IRIF in lymphocytes and will be compared with the dose estimated by physical dosimetric method to verify the feasibility of assessment of biological dose specific to α-emitters. Biological dosimetric information has a valuable role in the triage, diagnosis and treatment of persons incorporated with alpha radionuclides after an accident, and is important for epidemiological studies of late stochastic diseases - i.e. cancer in internal contaminated people. Furthermore, this persistent IRIF will become a new potential biomarker for health monitor of radiation workers.

核事故、核战争和核恐怖活动等会导致人体α核素内污染，而且环境核素如氡及其子体α粒子内照射对人类的致癌效应倍受人们关注，其内照射生物剂量估算问题亟待解决。本项目采用免疫荧光技术和细胞流式技术，从表观遗传学上研究人外周血淋巴细胞由DSB-集簇性损伤形成的"染色质结构变化标志物IRIF"对高LET α粒子辐射品质的特异性，建立Am-241源和氡及其子体α粒子离体照射人外周血淋巴细胞的剂量-效应曲线，并比较不同能量α粒子生物效应的差别；采用氡及其子体染毒活体动物，探索α核素在体内不均匀分布情况下外周血淋巴细胞和红骨髓细胞生物剂量之间的关系并引入修正因子；试用于氡及其子体暴露的矿工进行红骨髓累积剂量估算，与物理剂量估算结果相比较，初步验证其作为高LET α粒子内照射红骨髓生物剂量估算的可行性，为大剂量α核素内照射事故人员的医学救治和低剂量α核素内照射致癌的流行病学研究提供生物剂量依据。

高LET α核素内照射严重危害人类健康，尤其是环境高氡及其子体暴露诱发白血病特别是儿童白血病的危险已引起国际社会的高度关注，但是，作为评价其健康危害重要基础的内照射生物剂量估计问题，迄今尚未找到有效的解决途径。本研究首先采用241Am源α粒子离体照射G0期人外周血淋巴细胞（HPBLs），应用免疫荧光技术检测发现，DNA双链断裂分子标志物γ-H2AX IRIF线性径迹这一特殊形态可作为高LET α粒子辐射品质特异性的生物指标，用于判断是否存在α粒子内照射。继而，本研究建立了适用于人体γ-H2AX辐射生物剂量计研究的大鼠模型，通过离体大鼠外周血淋巴细胞（PBLs）和骨髓淋巴细胞（BMLs）氡暴露的体外试验和大鼠氡暴露的整体实验，进一步证实线性γ-H2AX IRIF径迹可作为判断是否存在高氡暴露的生物标志物，同时建立了体外0-5.86mGy氡暴露诱发大鼠PBLs和BMLs γ-H2AX IRIF的剂量-效应曲线，证实了γ-H2AX IRIF剂量-效应在体外氡暴露大鼠PBLs和BMLs中的一致性，确定了氡吸入大鼠BMLs/PBLs吸收剂量的比值作为红骨髓剂量估算修正因子，由此建立了通过检测PBLs γ-H2AX IRIF产额估算氡吸入大鼠红骨髓生物剂量的估算方法，并为采用物理剂量估计方法依据红骨髓生物剂量值估算其他组织器官的吸收剂量提供重要参数。在此基础上，本研究进一步建立了0-6.85mGy氡暴露诱发HPBLs γ-H2AX IRIF的线性剂量-效应曲线，证实γ-H2AX IRIF比53BP1和p-KAP-1 IRIF具有更高的灵敏度，其能估算的最低剂量可达1.74mGy，而且在停止氡暴露的HPBLs中持续存在12h，是估算慢性累积低剂量氡暴露生物剂量的良好指标。研究还意外发现，X射线和α粒子照射可诱发HPBLs形成代表凋亡的全核γ-H2AX反应；ATM/DNA-PKcs抑制剂、p53抑制剂和Pan-caspase抑制剂均能显著降低X射线照射诱导的全核γ-H2AX反应和细胞凋亡，而抑制Pan-caspase因未加重HPBLs的基因组不稳定性可能是更有发展前景的防护HPBLs辐射损伤的药理途径。. 本研究首次建立的γ-H2AX IRIF检测用于判断是否存在高氡暴露和估算红骨髓生物剂量的方法，对于早期监测和评价高氡暴露对人类的健康危害具有重要的研究意义和应用前景