电离辐射诱导的DNA双链断裂

利用γ射线和不同ＬＥＴ的碳离子幅照小鼠Ｂ１６黑色素瘤细胞、Ｈｅｌａ细胞、Ｖ７９中国仓鼠肺细胞和人肝癌ＳＭＭＣ－７７２１细胞的ＤＮＡ,采用脉冲场凝电泳结合荧光扫描技术研究了ＤＮＡ双链断裂（ＤＳＢ）片段的分布。结果发现ＤＳＢ片段是非随机分布的,而且这种分布与ＤＮＡ序列有关。原因可能在于沉积的能量直接或间接沿ＤＮＡ链迁移,链上相对较弱的化学键优先产生反应,并最终导致链的断裂,从而引起断裂的不均匀分布。     

渥曼青霉素对端粒DNA链断裂重连接作用的影响

端粒是位于真核细胞染色体末端的DNA-蛋白质复合体,在维持染色体稳定上起着重要的作用,并且与细胞的衰老、癌变有着密切的关系。本实验观察了DNA依赖性蛋白激酶(DNA-dependent protein kinase,DNA-PK)抑制剂-渥曼青霉素(wortmannin,WM)对H2O2诱导的HeLa细胞端粒DNA链断裂重连接效应。结果表明WM能够显著地抑制H2O2诱导的HeLa细胞端粒DNA链断裂后的重连接作用,提示DNA-PK参与了端粒DNA链断裂损伤的修复过程。     

3-氨基苯酰胺对氧化应激诱导的HeLa细胞端粒DNA链断裂重连接作用的影响

端粒是位于真核细胞染色体末端的DNA-蛋白质复合体,在维持染色体稳定上起着重要的作用,并且与细胞的衰老和凋亡有着密切的关系.在各种DNA损伤中,单链断裂(single-strand breaks, SSBs)是最常见的类型之一,既可直接通过内源活性氧或离子化辐射产生,也可间接地在DNA代谢或碱基切除修复期间产生.已知多聚(ADP-核糖)聚合酶［poly(ADPribose) polymerase, PARP］在SSBs修复中起着极为重要的作用.本实验观察了PARP抑制剂3-氨基苯酰胺(3-aminobenzamide, 3-AB)对氧化应激诱导的HeLa细胞端粒DNA链断裂重连接的效应以及对过氧化氢(H2O2)抑制HeLa细胞增殖的影响.结果表明3-AB能够显著地抑制氧化应激诱导的HeLa细胞端粒DNA链断裂后的重连接作用,并能增强H2O2对HeLa细胞增殖的抑制作用,提示PARP参与了端粒DNA链断裂损伤的修复过程.     

电离辐射诱导基因的研究进展

电离辐射诱导基因是一类受电离辐射调控表达的基因,其表达随辐射条件和所处生理环境的不同呈现复杂多变的特征。电离辐射诱导基因参与细胞内各种代谢途径,在细胞周期调控、细胞生长调节、细胞凋亡、DNA损伤修复中发挥着重要的作用。介绍了电离辐射诱导基因的种类、功能,及其引起的生物效应的分子机制及应用。     

用脉冲电场凝胶电泳检测电离辐射所致哺乳动物细胞DNA双链断裂

 本文将反向交变电场和六角形电极电场这两种脉冲电场凝胶电泳技术应用于X线照射小鼠乳癌细胞SR-1所致DNA双链断裂的检测,在本实验条件下,用这种电泳都能检测到低至1.5Gy照射所产生的DNA双链断裂,并且用六角形电极电场电泳获得了DNA双链断裂程度与照射剂量之间的良好线性关系,此外,还用此方法观察了不同浓度自由基清除剂DMSO对X线照射SR-1细胞所致DNA双链断裂的保护作用,结果进一步证实本方法的可靠性。     

DNA链断裂检测技术的进展

DNA链损伤特别是DNA双链段裂(dsb)的检测方法是研究DNA辐射损伤的一个关键因素.已发展的检测DNA dsb的方法很多,但各种检测法均有其一定的优越性和适用范围,近年来应用较多并日益受到重视的新方法有原位杂交法,彗星试验(单细胞电泳法)以及高效毛细管电泳法等等.     

激光对DNA的作用

简述了激光对DNA的作用。介绍了与激光辐照导致DNA链断裂、光产物形成及其分子机理、激光损伤与突变的关系以及高能脉冲激光和双光子作用有关的一些研究结果。     

γ射线诱导DNA链断裂的机制研究

通过凝胶电泳扫描法测试了γ射线辐射下ｐＢＲ３２２超螺旋ＤＮＡ的单链断裂（ＳＳＢ）和双链断裂（ＤＳＢ）与辐射剂量的关系,验证了ＳＳＢ的产生方式,发现ＤＳＢ的形成与ＤＮＡ溶液中自由基清除效能σ有关：一方面,αＤＳＢ与βＤＳＢ的比随σ的增大而非线性增大;另一方面,σ＜１０８ｓ－１时,αＤＳＢ可由单自由基传递机制产生,而σ＞２×１０８ｓ－１时,αＤＳＢ则主要通过ＬＭＤＳ机制形成。另外,发现组成βＤＳＢ的两互补链上ＳＳＢ的距离ａ几乎不受自由基清除效能的影响。     

ADP-核糖基转移酶的抑制对γ线所致肿瘤细胞DNA损伤的加强作用

本文探讨了ADP—核糖基转移酶(ADPRT)的活性、DNA单链断裂(SSB)重接和细胞潜在致死质损伤修复(PLDR)三者的关系。证明了ADPRT的特异性抑制剂3—氨基苯甲酰胺(3AB)能阻抑γ线所致的小鼠腹水瘤细胞DNA SSB的重接和PLDR。为增强放射治疗的效果提供了可能的新途径。     

Interaction of Microwaves and a Temporally Incoherent Magnetic Field on Single and Double DNA Strand Breaks in Rat Brain Cells

The effect of a temporally incoherent magnetic field noise on microwave-induced DNA single and double strand breaks in rat brain cells was investigated. Four treatment groups of rats were studied: microwave-exposure (continuous-wave 2450-MHz microwaves, power density 1 mW/cm², average whole-body specific absorption rate of 0.6 W/kg), noise-exposure (45 mG), microwave + noise-exposure, and sham-exposure. Animals were exposed to these conditions for 2h. DNA single- and double-strand breaks in brain cells of these animals were assayed 4h later using a microgel electrophoresis assay. Results show that brain cells of microwave-exposed rats had significantly higher levels of DNA single- and double-strand breaks when compared with sham-exposed animals. Exposure to noise alone did not significantly affect the levels (i.e., they were similar to those of the sham-exposed rats). However, simultaneous noise exposure blocked microwave-induced increases in DNA strand breaks. These data indicate that simultaneous exposure to a temporally incoherent magnetic field could block microwave-induced DNA damage in brain cells of the rat.     

Histochemical Demonstration of DNA Double Strand Breaks by in Situ 3'-tailing Reaction in Apoptotic Endometrium

A new histochemical technique, called in situ 3'-tailing reaction (ISTR), to detect DNA double strand breaks (DSB) was developed and applied to tissue sections of apoptotic endometrium. To demonstrate DSB, biotin-labeled and unlabeled dATPs with terminal deoxynucleotidyl transferase (TdT) were added to the many 3-hydroxyl termini of DNA fragments generated in the apoptotic cells. For an efficient 3'-end labeling, it was necessary to treat the sections with λ-exonuclease (λEx) prior to the TdT reaction to generate 3'-protruding ends. The λEx-TdT reaction specifically labeled nuclear fragments in the apoptotic cells in paraformaldehyde fixed frozen sections. In paraffin sections, pretreatment with proteinase K was effective for 3'-tailing reaction. ISTR should be a useful tool for detecting dying cells in both physiological and pathological states.     

衰老过程中大白鼠脾细胞的DNA单链断裂与重接能力的变化

 DNA被紫外线损伤后,由DNA切除修复酶切除嘧啶二聚体,随之以另一条正常的DNA链为模板修复合成DNA片段,最后由DNA连接酶将新合成的DNA片与原有的DNA链连接。本文用荧光法测定DNA修复过程中DNA单链的断裂及重接能力与衰老的关系。结果表明,不同年龄大鼠脾细胞均具有修复DNA单链断裂的能力,DNA单链断裂重接的能力与年龄有相关性,断乳鼠及青年鼠的脾细胞当保温至30min时,即开始了DNA链的重接,保温90min后则恢复到原有水平;而老年鼠脾细胞保温至90min时才开始DNA链的重接,保温150min,尚未恢复到原有水平。还发现,断乳鼠及老年鼠脾细胞的单链DNA含量高于青年鼠。     

DNA Strand Breaks in Mitotic Germ Cells of Caenorhabditis elegans Evaluated by Comet Assay

DNA damage responses are important for the maintenance of genome stability and the survival of organisms. Such responses are activated in the presence of DNA damage and lead to cell cycle arrest, apoptosis, and DNA repair. In Caenorhabditis elegans, double-strand breaks induced by DNA damaging agents have been detected indirectly by antibodies against DSB recognizing proteins. In this study we used a comet assay to detect DNA strand breaks and to measure the elimination of DNA strand breaks in mitotic germline nuclei of C. elegans. We found that C. elegans brc-1 mutants were more sensitive to ionizing radiation and camptothecin than the N2 wild-type strain and repaired DNA strand breaks less efficiently than N2. This study is the first demonstration of direct measurement of DNA strand breaks in mitotic germline nuclei of C. elegans. This newly developed assay can be applied to detect DNA strand breaks in different C. elegans mutants that are sensitive to DNA damaging agents.     

三硝基甲苯致晶状体上皮细胞DNA损伤作用的研究

本文通过出生后７天和２４个月两组大鼠晶状体与２０％三硝基甲苯（ＴＮＴ）甘油：水（８：２）混悬液体外培养,培养箱通５％ＣＯ２,恒温３７℃,观察ＴＮＴ对晶状体上皮细胞ＤＮＡ的损伤程度,结果以反映单链ＤＮＡ断裂强度的Ｆ６３０／Ｆ５３０比值表示。ＨＰＬＣ分析发现经体外ＴＮＴ孵育的晶状体内含有ＴＮＴ及其代谢产物;当共同培养９６ｈ后,Ｆ６４０／Ｆ５３０比值随着ＴＮＴ剂量增加而增大,达到４０μＬ（１１．７４μｍｏｌ／Ｌ）时趋向平稳;当用ＴＮＴ浓度为４．４０μｍｏｌ／Ｌ的相同剂量培养时,４８小时后单链ＤＮＡ断裂最为严重Ｆ６３０／Ｆ５３０比值为１．５０,明显高子正常对照组（Ｐ＜０．０１）;幼鼠单链ＤＮＡ断裂程度显著高于老龄鼠（Ｐ＜０．０１）。     

DNA Single Strand Breaks by Aromatic Nitroso Compounds in the Presence of Thiols

Aromatic nitroso compounds, nitrosobenzene (NB), N, N-dimethyl-4-nitrosoaniline (DMNA) and 3,5-dibromo-4-nitrosobenzene sulfonate (DBNBS), caused DNA single strand breaks in the presence of thiol compounds. The strand breaking was inhibited completely by free radical scavenger ethanol. Electron spin resonance (ESR) studies showed that hydronitroxyl (or sulfur-substituted nitroxyl) radicals were generated in the early stage of the interactions. Formation of these radicals was not inhibited by ethanol, indicating that these radicals did not directly contribute to the strand breaking. The DNA strand breaking was inhibited partially by superoxide dismutase and catalase under the limited conditions, but not by removal of oxygen from or addition of metal chelators to the reaction mixture. By ESR-spin trapping technique using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), the DMPO-OH spin adduct was detected. Formation of the spin adduct was inhibited by superoxide dismutase and catalase. The hydronitroxyl (or the sulfur-substituted nitroxyl) radicals may reduce oxygen into active oxygen species and also transformed by themselves into other unidentified free radical species to cause the DNA strand breaks.     

Early Detection of DNA Strand Breaks in the Brain After Transient Focal Ischemia: Implications for the Role of DNA Damage in Apoptosis and Neuronal Cell Death

Abstract: Using in situ DNA polymerase I-mediated biotin-dATP nick-translation (PANT) and terminal deoxynucleotidyl-transferase-mediated dUTP nick end-labeling (TUNEL), we investigated the evolution of DNA strand breaks, a marker of DNA damage, in rat brain after 1 h of middle cerebral artery occlusion and various durations of reperfusion. DNA single-strand breaks (SSBs) detected by PANT were present in neurons after as little as 1 min of reperfusion. Numbers of neurons containing an SSB increased progressively in the ischemic core but decreased in the ischemic penumbra after 1 h of reperfusion. DNA double-strand breaks (DSBs) detected by TUNEL were first seen in neurons after 1 h of reperfusion, and their numbers then increased progressively in the ischemic core, with a regional distribution similar to that of SSBs. However, the number of SSB-containing cells was greater than that of DSB-containing cells at all time points tested. SSB-containing cells detected within the first hour of reperfusion were exclusively neuronal and exhibited normal nuclear morphology. At 16–72 h of reperfusion, many SSB- and DSB-containing cells, including both neurons and astrocytes, showed morphological changes consistent with apoptosis. Gel electrophoresis of DNA isolated from the ischemic core showed DNA fragmentation at 24 h, when both SSBs and DSBs were present, but not at 1 h, when few DSBs were detected. These results suggest that damage to nuclear DNA is an early event after neuronal ischemia and that the accumulation of unrepaired DNA SSBs may contribute to delayed ischemic neuronal death, perhaps by triggering apoptosis.