高LET辐射致DNA和生物体损伤的机制及其防护研究
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摘要
脱氧核糖核酸(DNA)是辐射生物学效应的最重要和灵敏的靶分子。电离辐射通过直接作用和自由基的间接作用可引起DNA分子多种类型的损伤，如碱基损伤、糖基损伤、单链和双链断裂以及DNA交联等。其中双链断裂(DSBs)是最关键的损伤类型，它的正确和完全修复可保证细胞的存活，而错误修复和残余DNA损伤将导致细胞的死亡、突变和转化。
重离子和质子是具有高传能线密度(LET)的辐射。与低LET辐射相比，其能量沉积密集，通过物质时有着完全不同结构的电离径迹，径迹结构复杂，能引起较高的相对生物学效应(RBE)。在辐射生物学实验中，具有高LET的粒子的使用，为研究不同离子密度和生物学系统之间的相互关系及证实模型和理论的物理基础提供了基本和良好的工具。研究高LET辐射与生物物质的相互作用对于重离子和质子在放射治癌中的应用具有十分重要的意义，同时对于暴露于高LET辐射情形下，如太空辐射及核辐射环境下人员的危险性评估和辐射防护也是十分必要的。
本研究利用原子力显微镜(AFM)技术和凝胶电泳方法，在分子水平上研究了具有高LET的重离子辐射中直接和间接作用致DNA链断裂的物理化学机理及自由基清除剂的辐射防护作用。利用光学显微镜手段，在细胞水平上观测了质子辐射诱发的一些生物学效应。以期为重离子和质子治癌、太空辐射和核辐射的危险性评估及其防护等提供有价值的实验依据。
选用HI-13串列加速器加速的具有不同LET值的~7Li和~(12)C重离子，以不同的剂量分别对干状及不含和含自由基清除剂的水溶液pUC19质粒DNA进行了辐照。使用AFM在纳米尺度上直接观测了辐射诱发的DNA碎片及分子形态的变化。得到了DNA碎片长度的分布和双链断裂数目随辐射剂量的变化规律，评估了自由基清除剂甘露醇和维生素C的抗辐射能力。同时，使用凝胶电泳方法分析了实验样品，对AFM的结果进行了验证和补充。研究结果表明：
对于水溶液pUC19质粒DNA，具有110keV／μm左右LET值的~7Li离子诱发的生物效应最为严重。与具有低LET的电子和高LET的中子(55keV／μm)相比，在相近剂量下，具有较高LET的~7Li离子可诱发DNA分子发生集团损伤，即形成更多和更短的DNA小碎片，从而使DSB的分布更局部和更密集；干状DNA及DNA水溶液浓度和自由基清除剂对DNA链断裂产额的影响均暗示，在水溶液环境下，~7Li离子辐射诱发的DNA链断裂是直接作用和自由基的间接作用的共同结果，而自由基的作用为主导因素；
在~7Li和~(12)C重离子辐射中，自由基清除剂甘露醇和维生素C均能与DNA分子竞争自由基，清除辐射过程中产生的自由基，有效地减少重离子诱发的DNA链断裂的产生，具有一定的重离子辐射防护能力：与γ辐射相比，自由基清除剂对重离子辐射诱发产生的DSB的清除能力略小，表明重离子辐射可诱发更多的不能清除的DSB，这些DSB主要由直接作用和局部多重损伤位点(LMDS)所引起；
质子辐射诱发ICR小鼠的皮肤及心肌、肝和肺等深层组织发生了病理性改变。这些结果为进一步深入研究质子辐射致生物体损伤机理提出了挑战。
总之，传统的核物理实验技术与先进的AFM技术和现代分子生物学技术的成功结合，为辐射生物损伤机制及其防护的研究提供了一种重要和有力的研究手段，可实现在分子水平上获得高LET辐射致DNA损伤及其防护的更为精细的信息和丰富的实验数据。
It is widely accepted that DNA is the crucial sensitive target of radiation. Numerous lesions including sugar alteration, base damage, single- and double-strand breaks together with cross-links have been identified in irradiated DNA. The DNA double strand breaks (DSBs) are considered as the most critical type of lesions. Correct and complete repair of DSBs guarantees cellular survival, while incorrect repair and residual DNA damage may lead to cell killing, mutation or transformation.
Heavy ions and protons with high linear energy transfer (LET) values can depose more energies and have different ionizing track structure while through the matter comparing with the low-LET radiation. Their complex ionizing tracks lead to multiple ionization events within nanometer regions of the target, resulting in the induction of complex damage and higher relative biological effectiveness (RBE). The use of high LET particles in radiobiological experiments provides a fundamental and powerful tool to study the relationship between different ionization densities and biological system as well as to prove the physical basis of models and theories. The knowledge of the interaction of high LET radiation with biological matter is of great importance for the application of heavier ions and protons therapy. It is also essential in radioprotection for estimation of risk to crews and patients in case of exposure to high LET radiation, such as space or nuclear radiation environment.
The goal of this study is to investigate the physical and chemical mechanism of DNA strand breaks induced by direct and indirect effect of heavy ions radiation with high LET and corresponding radioprotection action of free radical scavenger by means of atomic force microscopy (AFM) and gel electrophoresis at the molecular level. In addition, the biological effects induced by protons are observed using the optical microscopy at the cellular level.
Choosing ~7Li and ~(12)C heavy ions with different LET accelerated by HI-13 tandem accelerator respectively, the dry and aqueous pUC19 plasmid DNA with or without free radical scavenger are irradiated with different doses in air. AFM is used to directly visualize the DNA fragments and the change of DNA form resulting from exposure to heavy ions radiation at the nanometer scale. The distributions of DNA fragment lengths and the changes of number of DSBs as the dose increase are obtained. The radioprotective capability of scavenger, mannitol and vitamin C is estimated. At the same time, the irradiated DNA samples are also analyzed by gel electrophoresis technique in order to prove and supplement experimental results obtained by AFM. The results are as follows,
There is a maximum biological effectiveness when the pUC19 plasmid DNA in aqueous solution is irradiated by ~7Li ions at a LET around 110keV/μm. Comparing with the low LET electron and high LET neutron irradiation, the higher LET ~7Li ions induce the cluster DSB lesions that is formation of much larger and much shorter small DNA fragments, and then the distribution of DSB more locally and more densely at comparable dose. The influence of dry DNA, the concentration of DNA solution and free radical scavenger on yielding DNA strand breaks suggest that the DNA strand breaks induced by ~7Li ions as the corporate result of direct effect and indirect effect of free radical, and the free radical effect may be the main factor.
In ~7Li and ~(12)C ions radiation, the scavenger, mannitol and vitamin C can compete free radical with DNA molecule and scavenge the free radical generated during radiolysis, then reduce the yields of DNA strand breaks induced by heavy ions efficiently, which suggest that they have stronger radioprotective capability against heavy ions radiation. The scavenging capacity of scavenger for heavy ions radiation induces DSB is lower than that one induced byγ-rays, namely the heavy ions radiation may induce a much number of DSBs that can not be scavenged, those DSBs mainly are produced by mechanism of direct effect and are induced by the locally multiply damaged sites (LMDS).
The protons radiation induces the ICR mouse' tissue, such as skin, cardiac muscle, lung and liver appear pathologic change, which results bring forward a challenge to further study of organism damage induced by protons radiation.
In conclusion, combined traditional nuclear physical experimental technique with advanced AFM and modern molecular biological technology successfully, an important and powerful means is provided for study of the mechanism of biological damage induced by radiation and corresponding radioprotection. Using of those means, the more delicate information and more abundant experimental data of DNA damage and its protection induced by high LET radiation can be obtained at molecular level.
重离子和质子是具有高传能线密度(LET)的辐射。与低LET辐射相比，其能量沉积密集，通过物质时有着完全不同结构的电离径迹，径迹结构复杂，能引起较高的相对生物学效应(RBE)。在辐射生物学实验中，具有高LET的粒子的使用，为研究不同离子密度和生物学系统之间的相互关系及证实模型和理论的物理基础提供了基本和良好的工具。研究高LET辐射与生物物质的相互作用对于重离子和质子在放射治癌中的应用具有十分重要的意义，同时对于暴露于高LET辐射情形下，如太空辐射及核辐射环境下人员的危险性评估和辐射防护也是十分必要的。
本研究利用原子力显微镜(AFM)技术和凝胶电泳方法，在分子水平上研究了具有高LET的重离子辐射中直接和间接作用致DNA链断裂的物理化学机理及自由基清除剂的辐射防护作用。利用光学显微镜手段，在细胞水平上观测了质子辐射诱发的一些生物学效应。以期为重离子和质子治癌、太空辐射和核辐射的危险性评估及其防护等提供有价值的实验依据。
选用HI-13串列加速器加速的具有不同LET值的~7Li和~(12)C重离子，以不同的剂量分别对干状及不含和含自由基清除剂的水溶液pUC19质粒DNA进行了辐照。使用AFM在纳米尺度上直接观测了辐射诱发的DNA碎片及分子形态的变化。得到了DNA碎片长度的分布和双链断裂数目随辐射剂量的变化规律，评估了自由基清除剂甘露醇和维生素C的抗辐射能力。同时，使用凝胶电泳方法分析了实验样品，对AFM的结果进行了验证和补充。研究结果表明：
对于水溶液pUC19质粒DNA，具有110keV／μm左右LET值的~7Li离子诱发的生物效应最为严重。与具有低LET的电子和高LET的中子(55keV／μm)相比，在相近剂量下，具有较高LET的~7Li离子可诱发DNA分子发生集团损伤，即形成更多和更短的DNA小碎片，从而使DSB的分布更局部和更密集；干状DNA及DNA水溶液浓度和自由基清除剂对DNA链断裂产额的影响均暗示，在水溶液环境下，~7Li离子辐射诱发的DNA链断裂是直接作用和自由基的间接作用的共同结果，而自由基的作用为主导因素；
在~7Li和~(12)C重离子辐射中，自由基清除剂甘露醇和维生素C均能与DNA分子竞争自由基，清除辐射过程中产生的自由基，有效地减少重离子诱发的DNA链断裂的产生，具有一定的重离子辐射防护能力：与γ辐射相比，自由基清除剂对重离子辐射诱发产生的DSB的清除能力略小，表明重离子辐射可诱发更多的不能清除的DSB，这些DSB主要由直接作用和局部多重损伤位点(LMDS)所引起；
质子辐射诱发ICR小鼠的皮肤及心肌、肝和肺等深层组织发生了病理性改变。这些结果为进一步深入研究质子辐射致生物体损伤机理提出了挑战。
总之，传统的核物理实验技术与先进的AFM技术和现代分子生物学技术的成功结合，为辐射生物损伤机制及其防护的研究提供了一种重要和有力的研究手段，可实现在分子水平上获得高LET辐射致DNA损伤及其防护的更为精细的信息和丰富的实验数据。
It is widely accepted that DNA is the crucial sensitive target of radiation. Numerous lesions including sugar alteration, base damage, single- and double-strand breaks together with cross-links have been identified in irradiated DNA. The DNA double strand breaks (DSBs) are considered as the most critical type of lesions. Correct and complete repair of DSBs guarantees cellular survival, while incorrect repair and residual DNA damage may lead to cell killing, mutation or transformation.
Heavy ions and protons with high linear energy transfer (LET) values can depose more energies and have different ionizing track structure while through the matter comparing with the low-LET radiation. Their complex ionizing tracks lead to multiple ionization events within nanometer regions of the target, resulting in the induction of complex damage and higher relative biological effectiveness (RBE). The use of high LET particles in radiobiological experiments provides a fundamental and powerful tool to study the relationship between different ionization densities and biological system as well as to prove the physical basis of models and theories. The knowledge of the interaction of high LET radiation with biological matter is of great importance for the application of heavier ions and protons therapy. It is also essential in radioprotection for estimation of risk to crews and patients in case of exposure to high LET radiation, such as space or nuclear radiation environment.
The goal of this study is to investigate the physical and chemical mechanism of DNA strand breaks induced by direct and indirect effect of heavy ions radiation with high LET and corresponding radioprotection action of free radical scavenger by means of atomic force microscopy (AFM) and gel electrophoresis at the molecular level. In addition, the biological effects induced by protons are observed using the optical microscopy at the cellular level.
Choosing ~7Li and ~(12)C heavy ions with different LET accelerated by HI-13 tandem accelerator respectively, the dry and aqueous pUC19 plasmid DNA with or without free radical scavenger are irradiated with different doses in air. AFM is used to directly visualize the DNA fragments and the change of DNA form resulting from exposure to heavy ions radiation at the nanometer scale. The distributions of DNA fragment lengths and the changes of number of DSBs as the dose increase are obtained. The radioprotective capability of scavenger, mannitol and vitamin C is estimated. At the same time, the irradiated DNA samples are also analyzed by gel electrophoresis technique in order to prove and supplement experimental results obtained by AFM. The results are as follows,
There is a maximum biological effectiveness when the pUC19 plasmid DNA in aqueous solution is irradiated by ~7Li ions at a LET around 110keV/μm. Comparing with the low LET electron and high LET neutron irradiation, the higher LET ~7Li ions induce the cluster DSB lesions that is formation of much larger and much shorter small DNA fragments, and then the distribution of DSB more locally and more densely at comparable dose. The influence of dry DNA, the concentration of DNA solution and free radical scavenger on yielding DNA strand breaks suggest that the DNA strand breaks induced by ~7Li ions as the corporate result of direct effect and indirect effect of free radical, and the free radical effect may be the main factor.
In ~7Li and ~(12)C ions radiation, the scavenger, mannitol and vitamin C can compete free radical with DNA molecule and scavenge the free radical generated during radiolysis, then reduce the yields of DNA strand breaks induced by heavy ions efficiently, which suggest that they have stronger radioprotective capability against heavy ions radiation. The scavenging capacity of scavenger for heavy ions radiation induces DSB is lower than that one induced byγ-rays, namely the heavy ions radiation may induce a much number of DSBs that can not be scavenged, those DSBs mainly are produced by mechanism of direct effect and are induced by the locally multiply damaged sites (LMDS).
The protons radiation induces the ICR mouse' tissue, such as skin, cardiac muscle, lung and liver appear pathologic change, which results bring forward a challenge to further study of organism damage induced by protons radiation.
In conclusion, combined traditional nuclear physical experimental technique with advanced AFM and modern molecular biological technology successfully, an important and powerful means is provided for study of the mechanism of biological damage induced by radiation and corresponding radioprotection. Using of those means, the more delicate information and more abundant experimental data of DNA damage and its protection induced by high LET radiation can be obtained at molecular level.
引文
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