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3-38 Analysis of Lethal Effect Mediated by Low Dose Irradiation Induced-Secreted factors in Glioma cells S. Wada a) , A. Baden a) , E. Nakagawa a) , T. Kakizaki a) , T. Funayama b) , T. Sakashita b) , Y. Kobayashi b) and N. Ito a) a) Department of Veterinary Medicine, Kitasato University, b) Radiation-Applied Biology Division, QuBS, JAEA So far, we clarified that low dose ion beam irradiation induced enhanced lethal effect and this phenomenon related with sphingomyelinase (SMase). In this study we investigated if the enhanced lethal effect induced by low dose ion beams was involved in secreted materials, so-called bystander effect. In medium transfer experiments surviving fraction by X-ray and carbon beam irradiation decreased and DNA damage by low dose X-ray irradiation was induced. On the other hand, these effects were inhibited by SMase inhibitor. This result indicates that bystander effect considerably contributes enhanced lethal effect induced by low dose radiation and SMase effects at the upstream of this signal transduction. 1. はじめにこれまでに低線量炭素線照射による生存率は、高線量域の生存曲線からの外挿値よりも低い値を示し、低線量炭素線照射でも高い細胞致死効果が得られることを明らかにし、この低線量炭素線照射による細胞致死効果の増強のメカニズには細胞膜応答分子のスフィンゴミエリナーゼが関与することを明らかにしてきた。さらに、放射線照射後に細胞内にラジカル種が産生されることとこのラジカル種の産生はスフィンゴミエリナーゼ阻害剤によって抑制されることも明らかにしてきた。しかしながら、この細胞致死効果の増強メカニズムが直接照射を受けた細胞に過剰なシグナル伝達が誘導されたためか、照射細胞からの何らかのシグナル伝達因子によって(バイスタンダー効果)細胞内に過剰なシグナル伝達が誘導されたかは明かでない。そこで、細胞致死効果の増強メカニズムの細胞外からのシグナル伝達因子の影響について解析した。特に、腫瘍細胞では細胞外からのシグナル伝達においてギャップジャンクションの様な細胞間伝達機構は破綻していることが多いため、細胞外に放出される液性因子の関する機構について解析を行った。 2.実験方法グリオーマ細胞を用い、AVF サイクロトロンによって加速された 220 MeV C 5+ (LET=108 keV/μm)を照射した。また、対照の放射線として X 線を用いた放射線応答も解析した。細胞外放出因子による細胞致死効果を解析するため、X 線および炭素線照射した細胞を 1 時間培養後、その培養液のみを回収し、非照射細胞に添加し 1 時間培養後に colony formation assay を用いて、細胞致死効果を算出した(培養液交換実験)。さらに、細胞外放出因子による細胞致死効果のスフィンゴミエリナーゼの関与を解析するため、スフィンゴミエリナーゼ阻害剤による細胞致死効果の解析も行った。また、低線量 X 線照射について細胞外放出因子による細胞障害を DNA2 本鎖切断について γH2AX のリン酸を観察することにより評価した。 3. 結果および考察低線量域での X 線および炭素線照射における細胞外放出因子による細胞致死効果を観察した(Fig. 1)。いずれの放射線においても、培養液交換によって生存率の有意な低下が観察された。さらに、照射細胞をスフィンゴミエリナーゼ処理したとき、非照射細胞に誘導さ JAEA-Review 2010-065 - 94 - れる細胞死が抑制され、生存率がコントロールレベルまで回復した。この結果は、いずれの放射線も低線量照射によって細胞外放出因子によって細胞死が誘導され、この細胞致死誘導機構にもスフィンゴミエリナーゼが関与することを示唆している。これまでに照射後にラジカル種の産生とそのラジカル種の産生にはスフィンゴミエリナーゼが関与したため、非照射細胞に生成される DNA 損傷を観察したとき(Fig. 2)、培地交換 90 分後に DNA 損傷が生成され、この DNA 損傷生成はスフィンゴミエリナーゼ阻害剤によって抑制された。これらのことから、低線量放射線照射による細胞致死効果増強機構にはいずれの放射線においてもバイスタンダー効果の寄与が大きいと考えられ、さらに、この機構にはスフィンゴミエリナーゼがシグナル伝達過程のかなり上流で関与することが推察された。 Fig. 1 Killing effect of media transfer from irradiated culture. DNA damage rate Fig. 2 DNA damage induction in non-irradiated culture by transferring media from X-ray irradiated culture.
3-39 Ion Beam Irradiation Has Different Influences on the Expression of p53 in Cultured Human Retinal Vascular Endothelial Cells Exposed to L-dopa among 20 Ne, 12 C and 4 He K. Akeo a,b) , T. Funayama c) , N. Hamada d) , Y. Kobayashi c) and Y. Akeo a) a) Akeo Eye Clinic, b) Department of Ophthalmology, Keio University School of Medicine, c) Radiation-Applied Biology Division, QuBS JAEA, d) Radiation Safety Research Center, Central Research Institute of Electric Power Industry Deficiency of L-dopa causes degeneration of the substantia nigra in the brain and L-dopa is used in the treatment of Parkinson’s disease. We proved that L-dopa produced NO and superoxide, and had the cytotoxic effects on the retinal pigment epithelial cells 1) . L-dopa injected into the vitreous of the rats dilated the vena in the ciliary body 2) . We wondered if RE cells could be exposed to oxidative stress by administrated L-dopa. We already 3) reported that oxidative stress such as hyperoxia augmented the cytotoxicity of the aortic endothelial cells by L-dopa. Glutathione peroxidase (GPX), a selenium-dependent and lipid peroxide-scavenging enzyme that effectively reduces lipid peroxides with the concomitant oxidation of glutathione is distributed in mitochondria 4) . Faucher et al. measured the expression of two bcl-2 family members, bax and bcl-2, in a human endothelial like cell-line overexpressing the organic hydroperoxide-scavenging enzyme GPX, in the absence of any apoptotic/oxidant stimulus, and showed that overexpressing an antioxidant gene such as GPX in endothelial cells is able to change the basal mRNA and protein bax levels without affecting those of p53 and bcl-2. This phenomenon could be useful to antiatherogenic therapies which use antioxidants with the aim of protecting the vascular wall against oxidative stress injury 5) . Exposure to L-dopa inhibited the expression of GPX in RE cells. Ion beam irradiations both of 4 He and 12 C decreased the expression more remarkably than 20 Ne. The expression of GPX in RE cells incubated with L-dopa decreased significantly after 8 h of exposure to 4 He, and after 4 and 24 h of exposure to 12 C. On the contrary, ion beam irradiation of 20 Ne increased the expression of GPX in RE cells incubated with L-dopa after 4 h of the exposure of the irradiation significantly. We considered the different accumulation of the energy irradiated at a point by various ions could be concerned with the effects on the expression GPX in RE cells incubated with L-dopa. Established human RE cells in vitro incubated with L-dopa (250 µM) for 2 h were exposed to ionization radiation that is induced by acceleration of the ionizing atom of 350 MeV 20 Ne, 220 MeV 12 C, and 50 MeV 4 He. We obtained the RE cells after 0, 4, 8, 24 h of the irradiation and extracted total cellular RNA to synthesize cDNA. We used the Primer3 website to design the primers for RT-PCR JAEA-Review 2010-065 - 95 - amplification of the cDNA of p53 and 18S RNA. The reactions were carried out at the following temperature: 95 ºC, for denaturation; 60 ºC, for annealing; and 72 ºC, for extension for 17–27 cycles. After mixing the cDNA, primer, and SYBR green, the expression of 18S RNA and p53 was measured using the LightCycler system. The technology of this system is extremely innovative and enables rapid and simultaneous evaluation PCR experiments. Fluorometric analysis of the formed PCR products was performed as a real-time measurement either continuously or at specifically defined time points during each PCR cycle. The expression of p53 in RE cells significantly was not influenced by only exposure to L-dopa, but increased just after the irradiation both of 350 MeV 20 Ne and 50 MeV 4 He in those cells incubated with L-dopa. Ion beam irradiation caused the induction of apoptosis in RE cells damaged by lipid peroxidation with L-dopa. References 1) K. Akeo et al., Pigment Cell Res. 13 (2000) 80. 2) S. A. Amaki et al., Pigment Cell Res. 14 (2001) 256. 3) K. Akeo et al., Exp. Eye Res. 49 (1989) 335. 4) K. Watanabe, Tran. Soc. Pathol. Jpn. 76 (1986) 39. 5) K. Faucher et al., Mol. Cell Biochem. 277 (2005) 81.
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JAEA-Review 2010-065 JAEA Takasaki
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JAEA-Review 2010-065 PREFACE This r
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JAEA-Review 2010-065 and pulsed hea
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JAEA-Review 2010-065 1-23 Studies o
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JAEA-Review 2010-065 3-24 Lethal Ef
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JAEA-Review 2010-065 4-07 Fabricati
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JAEA-Review 2010-065 5. Status of I
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JAEA-Review 2010-065 1-13 Alpha-rad
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1-01 Radiation Resistance of InGaP/
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1-03 Evaluation of Soft Error Rates
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1-05 Heavy-ion Induced Current in S
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Total Ionizing Dose Tolerance of Si
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1-09 Evaluation of Single Event Eff
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Hydrogen Diffusion in a-Si:H Thin F
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1-13 Alpha-radiolysis of Organic Ex
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Study on Stability of Cs·Sr Solven
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Irradiation Effect of Gamma-Rays on
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1-19 Development of Radiation Resis
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1-21 Alpha-Ray Irradiation Damage o
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1-23 Studies on Microstructure and
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1-25 Effect of Groundwater Radiolys
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1-27 Simulation of Neutron Damage M
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1-29 Irradiation Hardening in Ion-i
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1-31 Preparation of Anion-Exchange
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1-33 Radiation-Induced Graft Polyme
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JAEA-Review 2010-065 2. Environment
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2-02 Development of Zwitterionic Mo
Page 60 and 61: Modification of Hydroxypropyl Cellu
Page 62 and 63: The Recovery of Precious Metals Usi
Page 64 and 65: 2-08 ESR Study on Carboxymethyl Chi
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Page 69 and 70: JAEA-Review 2010-065 3-28 Electron
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Page 74 and 75: 3-02 Mutagenic effects of He ion pa
Page 76 and 77: 3-04 Functional Analysis of Flavono
Page 78 and 79: 3-06 Generation New Ornamental Plan
Page 80 and 81: 3-08 Stability of Flower-colour Mut
Page 82 and 83: 3-10 JAEA-Review 2010-065 Effects o
Page 84 and 85: 3-12 Producing New Gene Resources i
Page 86 and 87: 3-14 Production of Soybean Mutants
Page 88 and 89: Assessment of Irradiation Treatment
Page 90 and 91: 3-18 Effect of Different LET Radiat
Page 92 and 93: 3-20 JAEA-Review 2010-065 Fungicide
Page 94 and 95: 3-22 JAEA-Review 2010-065 FACS-base
Page 96 and 97: 3-24 Lethal Effects of Different LE
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Page 100 and 101: 3-28 Electron Spin Relaxation Behav
Page 102 and 103: 3-30 Target Irradiation of Individu
Page 104 and 105: 3-32 Carbon-ion Microbeam Induces B
Page 106 and 107: 3-34 Biological Effects of Carbon I
Page 108 and 109: 3-36 Difference in Bystander Lethal
Page 112 and 113: 3-40 Irradiation with Carbon Ion Be
Page 114 and 115: 3-42 Expression of Two Gelsolins in
Page 116 and 117: 3-44 Analysis of Bystander Cell Sig
Page 118 and 119: 3-46 Quantitative Evaluation of Ric
Page 120 and 121: 3-48 Visualization of 107 Cd Accumu
Page 122 and 123: 3-50 Uniformity Measurement of Newl
Page 124 and 125: 3-52 Imaging and Biodistribution of
Page 126 and 127: 3-54 Improvement of Spatial Resolut
Page 128 and 129: 3-56 The Optimum Conditions in the
Page 130 and 131: 3-58 Evaluation of Cisplatin Concen
Page 132 and 133: 3-60 JAEA-Review 2010-065 Analysis
Page 134 and 135: 3-62 JAEA-Review 2010-065 Sensitivi
Page 136 and 137: JAEA-Review 2010-065 4-14 The Effec
Page 138 and 139: JAEA-Review 2010-065 4-39 Relations
Page 141 and 142: 4-01 Hydrogen Gasochromism of WO3 F
Page 143 and 144: 4-03 Synthesis of Single-Crystallin
Page 145 and 146: 4-05 Synergy Effects in Electron/Io
Page 147 and 148: Fabrication of Diluted Magnetic Sem
Page 149 and 150: 4-09 Gas Permeation Characteristics
Page 151 and 152: 4-11 Control of Radial Size of Poly
Page 153 and 154: 4-13 Behavior of N Atoms in Nitridi
Page 155 and 156: 4-15 JAEA-Review 2010-065 Annealing
Page 157 and 158: Low Temperature Ion Channeling of F
Page 159 and 160: 4-19 Radiation-Induced Electrical D
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4-21 Study on Cu Precipitation in E
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4-23 Evaluation of Fluorescence Mat
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4-25 Evaluation of the ZrC Layer fo
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4-27 Structure Analysis of K/Si(111
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4-29 LET Effect on the Radiation In
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4-31 Stabilization of Measurement S
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Systematic Measurement of Neutron a
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4-35 Measurement of Neutron Fluence
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4-37 Evaluation of the Response Cha
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4-39 Relationship between Internucl
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4-41 Analysis of Radiation Damage a
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4-43 Positive Secondary Ion Emissio
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4-45 JAEA-Review 2010-065 Ion Induc
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4-47 Fabrication of Dielectrophoret
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4-49 Fast Single-Ion Hit System for
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4-51 Development of Beam Generation
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JAEA-Review 2010-065 5. Status of I
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Operation of the AVF Cyclotron T. N
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Operation of Electron Accelerator a
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5-06 FACILITY USE PROGRAM in Takasa
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5-08 Radioactive Waste Management i
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Appendix 2 List of Related Patents
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Appendix 4 Type of Research Collabo
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表1.SI 基本単位基本量 SI
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