α、γ射线联合照射剂量效应与新型蛋白生物剂量计研究

发布时间：2018-04-08 22:31

本文选题：α粒子　切入点：γ射线　出处：《复旦大学》2012年硕士论文

【摘要】：随着核能和核科学技术的发展和广泛应用,电离辐射对生态环境和人类健康的影响越来越受到重视。a粒子是环境和职业暴露中的一种重要射线,除室内氡所产生的低剂量α辐射外,核电站反应堆事故所导致的α粒子照射将可能对公众产生显著的辐射损伤。在核事故中常常存在各种射线的联合照射,包括不同比例的a和γ射线。另外,放射性同位素所产生的α射线在核医学肿瘤治疗中的应用也日益广泛。由于α粒子具有较高的传能线密度(LET)和较强的相对生物学效应,它对人体健康的影响一直是放射生物学研究领域中最活跃的前沿之一。人体造血系统是具有较高辐射敏感性的组织之一,而微核是辐射损伤的敏感指标。本研究采用淋巴细胞检测分析了受a粒子、γ射线以及不同比例射线联合照射后细胞微核率的剂量效应,检测了两种射线照射间隔时间对微核形成的影响。通过本研究,以期为不同类型射线联合照射的生物剂量评价及辐射防护提供新的实验依据。生物剂量计是物理剂量的有效补充,为了研究适用于高通量快速分析的生物剂量计,本文还初步探讨了蛋白分子γ-H2AX.CDKN1A作为生物剂量计的可能性。第一部分：α粒子、丫射线联合照射剂量效应研究目的探讨α粒子、γ射线单独及联合照射引起淋巴细胞微核率的剂量效应,分析不同LET射线是否存在适应性反应。方法采用人B淋巴母细胞HMy2.CIR(简称为HMy),分别按以下条件进行辐射：①以0.1、0.25、0.5、0.75、1、2、3、4、5Gy的γ射线照射；②以0.01、0.025、0.05、0.1、0.15、0.2、0.5、0.75和1Gy的241Ama粒子照射；为了对细胞进行α照射,将淋巴细胞培养于自制的以3μm Mylar膜为底的培养皿中,Mylar膜预先以相对分子质量为150-300kd的多聚赖氨酸包被过夜,使得淋巴细胞能够贴壁生长。③对于联合照射,先采用0.025、0.1、0.2、0.5Gy的α粒子照射,然后立刻以0.25、0.75、2、3和4Gy的γ射线进行照射；或者先采用0.25、0.75、1、2Gy的γ射线后,然后立刻以0.025、0.1、0.2、0.5Gy的α粒子进行照射；④0.025、0.1Gy的α粒子照射后4h给予2Gy的γ射线,或0.1Gyγ射线照射后4h给予0.2、0.5a粒子照射。用细胞分裂阻滞法(CB法)检测细胞微核发生率。结果①对γ射线照射,微核率剂量效应符合线性平方模型Y=c+aD+βD2；②对α粒子照射,当照射剂量小于0.25Gy时,细胞微核率随剂量的增加线性增加,当其剂量进一步增加时,微核率剂量效应曲线呈现下弓型,可以采用反映辐射旁效应的BaD模型Y=c+αD+σ(1-exp(-δD))exp(-βD)进行很好的拟合。③对于联合照射,先照射α粒子时,当α粒子剂量较低时,微核率的剂量效应与Y射线照射时的相似；但当α粒子剂量较大时,微核率的剂量效应则更接近于a粒子照射。同时,0.2、0.5Gyα粒子照射联合γ射线照射引起的微核率显著高于单独照射时的微核率之和(t=5.22～11.86,P0.01)。先照射γ射线时,给予小剂量γ射线0.25Gy后立刻给予0.2、0.5Gy的α粒子,细胞微核率显著高于单独照射时的微核率之和(t=1.92,0.30,P0.01)；但当先给予0.75、2Gy γ射线后再照射0.2Gyα粒子时,微核率与单独照射时微核率之和无显著差别,而给予0.5Gy α粒子显示引起的微核率高于单独照射时的微核率之和(t=2.79,4.42,P0.05),且有统计学意义。一般说来,先照射α粒子的联合照射引起的微核率高于先照射γ射线的联合照射。④对于适应性试验,小剂量α粒子照射后Oh与4h给予γ射线照射,所引起的微核率无差异；而先给予0.1Gyγ射线,4h后给予α粒子照射,可以降低细胞微核率(P0.05)。结论α粒子照射具有与γ射线不同的辐射损伤规律,辐射旁效应可能在其中发挥了重要作用。对于先α粒子后γ射线的联合照射可以协同增强辐射损伤：而先γ射线后α粒子的联合辐射,当α射线剂量小于0.2Gy时,联合照射表现为拮抗效应；当α射线剂量大于0.2Gy时,联合照射产生协同效应。另外,当两次照射之间具有适当的时间间隔时,小剂量的γ射线可诱发辐射适应性反应,而小剂量的a粒子则未出现适应性反应。第二部分；新型蛋白生物剂量计探讨目的探讨DNA损伤修复相关蛋白γ-H2AX、CDKNIA作为生物剂量计的可能性。方法人B淋巴母细胞HMy2.CIR(HMy)给予一定剂量α粒子或γ射线照射后,分析γ-H2AX及CDKN1A的时间响应关系,获知表达量较高的时间点,并在此时间点上,以流式细胞仪检测受0.01-1Gy α粒子或0.1-5Gyγ射线照射后,γ-K2A、CDKNIA蛋白表达量的剂量效应。结果①经过α粒子和γ射线照射后,细胞γ-H2AX蛋白表达的第一次峰值分别出现在照射后0.5h和2h；对这两种辐射,γ-H2AX蛋白在照射后24h时仍有较显著表达；经不同剂量α粒子、γ射线照射后,γ-H2AX蛋白剂量效应具有一定的规律性。②经过α粒子辐射后,细胞CDKN1A蛋白表达的第一次峰值出现在照射后3h,在照射后24h出现第二次高表达；Y射线照射后此蛋白的表达在照射后24h出现了增高。结论不同LET辐射后,细胞γ-H2AX蛋白和CDKN1A蛋白表达有一定的时间响应规律,γ-H2AX蛋白表达具有一定的剂量效应。
[Abstract]:With the development of nuclear energy and nuclear science and technology and wide application of ionizing radiation on the ecological environment and human health has been paid more and more attention of.A particles is an important environment and occupation in the X-ray exposure, in addition to low doses of radiation generated by the indoor radon alpha, alpha particle irradiation in reactor of nuclear power plant accident caused by may the radiation produced significant damages to the public. There are often various rays combined with radiation in nuclear accident, including the different proportion of a and gamma ray. In addition, the application of alpha ray isotope produced in nuclear medicine in the treatment of cancer is increasing. The alpha particle has high linear energy transfer (LET) and strong relative biological effect, its impact on human health has been one of the most active research areas in radiation biology. The human hematopoietic system with high radiation sensitivity of a tissue, The micronucleus test is a sensitive indicator of radiation damage. This study uses lymphocytes analyzed by a particles, gamma ray dose effect and micronucleus rate of different proportion of X-ray combined with irradiation, detected the effects of two irradiation time interval on micronucleus formation. Through this study, in order to provide new experimental evidence for biological dose evaluation of different types of radiation combined with radiation and radiation protection.
Biological dosimeter is an effective supplement to physical dose. In order to study the biological dose meter suitable for high-throughput and fast analysis, we also probed into the possibility of protein molecule gamma -H2AX.CDKN1A as a biological dosimeter.
The first part: A Study on the dose effect of alpha particle and joint irradiation
Objective to investigate the dose effect of alpha particles, gamma ray alone and combined irradiation on the micronucleus rate of lymphocyte, and to analyze the adaptive response of different LET rays.
Methods using human B lymphoblastoid cell line HMy2.CIR (HMy), respectively according to the following conditions: 1. The radiation of gamma ray irradiation on 241Ama 0.1,0.25,0.5,0.75,1,2,3,4,5Gy; the 0.01,0.025,0.05,0.1,0.15,0.2,0.5,0.75 and 1Gy particles; for Zhaoshe to cell culture dish will be in lymphocyte culture based on 3 m Mylar film as the bottom of the self-made. Mylar film in advance with a relative molecular weight of 150-300kd poly-L-lysine overnight, making lymphocytes adherent. The combined irradiation, first using 0.025,0.1,0.2,0.5Gy's alpha particle irradiation, then immediately irradiated with gamma ray 0.25,0.75,2,3 and 4Gy; or by 0.25,0.75,1,2Gy gamma rays, and then immediately exposure to alpha particles of 0.025,0.1,0.2,0.5Gy; the 0.025,0.1Gy alpha particle irradiation after gamma ray 4H giving 2Gy, 0.1Gy or gamma irradiation 4h give 0.2,0.5a particle irradiation. Split block method (CB method) using cell micronucleus rate were detected. Results of gamma ray irradiation, the micronucleus rate of dose effect consistent with the linear quadratic model of Y=c+aD+ beta D2; the alpha particle irradiation, when the dose is less than 0.25Gy, the micronucleus rate increased linearly with the increase of dose, when the dose was further increased, the micronucleus rate showed a dose effect curve under the arch type, can be used to reflect the bystander effect of BaD model Y=c+ D+ (1-exp (- alpha sigma delta D)) exp (beta D) fitted very well. The first alpha particle irradiation combined with irradiation, when the dose of alpha particles when low dose Y irradiation effect and micronucleus rate is similar; but when alpha particles at high doses, dose effect on micronucleus rate is more close to the a particle irradiation. At the same time, 0.2,0.5Gy induced by alpha particle irradiation combined with gamma ray irradiation of the micronucleus rate was significantly higher than those of single irradiation The micronucleus rate and (t=5.22 - 11.86, P0.01). The first gamma rays and alpha particles giving small doses of gamma ray 0.25Gy after 0.2,0.5Gy immediately, the micronucleus rate of micronucleus rate was significantly higher than that of single irradiation and the (t=1.92,0.30, P0.01); but first give 0.75,2Gy gamma ray irradiation after 0.2Gy alpha the particle, the micronucleus rate and micronucleus rate of single irradiation and no significant difference, but given the 0.5Gy alpha particle display micronucleus rate induced micronuclei rate was higher than that of single irradiation and the (t=2.79,4.42, P0.05), and the difference was statistically significant. In general, combined with radiation caused by irradiation of alpha particle irradiation to the micronucleus rate higher than the first gamma rays. 4 for adaptability test, small dose of alpha particle irradiation after Oh and 4H treated with gamma irradiation, caused by the micronucleus rate had no difference; and give the 0.1Gy ray, 4H treated with alpha particles irradiation, can reduce the micronucleus rate (P0.05).
Conclusion alpha particle irradiation with radiation injury and regularity of different gamma ray radiation, bystander effect may play an important role in them. The first alpha particles combined with gamma rays can enhance radiation damage: first gamma ray after alpha particles combined with radiation, when alpha radiation dose is less than 0.2Gy, combined with radiation performance antagonistic effect; when the alpha dose is more than 0.2Gy, combined with radiation synergy. In addition, when has the proper time interval between the two irradiation, gamma ray radiation can induce a small dose of adaptive response, while the a particles in small doses did not show adaptive responses.
The second part of the new protein biological dosimeter
Objective to investigate the possibility of DNA damage repair related protein gamma -H2AX and CDKNIA as a biological dosimeter.
Methods human B lymphoblastoid cell line HMy2.CIR (HMy) to give a certain dose of alpha particle or gamma ray irradiation, gamma -H2AX and CDKN1A analysis of the time response relationship, get a higher expression of the time, and this time, flow cytometry was used to detect 0.01-1Gy alpha particles or 0.1-5Gy gamma ray irradiation, gamma -K2A, CDKNIA protein expression dose amount.
Results after the alpha particle and gamma ray irradiation, the first peak expression of -H2AX cells appeared 0.5h in gamma irradiation and 2H; on the two kinds of gamma radiation, there is still a significant expression of -H2AX protein in 24h after irradiation; with different doses of alpha particles, gamma irradiation, gamma dose -H2AX protein the effect has a certain regularity. After the alpha particle radiation, the first peak cell CDKN1A expression in 3h after irradiation, second high expression appeared at 24h after irradiation; Y after irradiation the expression of this protein in irradiated 24h had increased.
Conclusion after different LET radiation, the expression of gamma -H2AX protein and CDKN1A protein has a certain time response law, and the expression of gamma -H2AX protein has a certain dose effect.

【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R814.2

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