TNT体外大鼠白内障模型中的DNA单链断裂及其重接修复的研究

在大鼠晶状体器官培养的条件下,运用单细胞电泳法（ＳＣＧ）,对远在晶状体混浊之前的晶状体上皮细胞进行了有关ＴＮＴ致其ＤＮＡ损伤（ＳＳＢ）与修复的初步观察。提示ＤＮＡ损伤也是体外ＴＮＴ性白内障中的早期变化。     

激光、血卟啉对肿瘤细胞DNA单链断裂及其重接的影响

用简化的Kohn氏碱洗脱法,观察了光敏剂血卟啉衍生物(HPD)对小鼠S-180肿瘤细胞DNA单链断裂及其重接修复的影响。激光HPD能导致S-180细胞DNA单链断裂明显增加,而且这种断裂随着保温时间的延长,继续增多。在本实验条件下没有观察到HPD对X线的增敏作用,HPD不能增加X线所致的DNA单链断裂,也不能影响其重接。单链断裂重接动力学的实验进一步证明了这个论点。     

TNT体外大鼠白内障模型中的DNA单链断裂及其重接修复的研究

在大鼠晶状体器官培养的条件下,运用单细胞电泳法（ＳＣＧ）,对远在晶状体混浊之前的晶状体上皮细胞进行了有关ＴＮＴ致其ＤＮＡ损伤（ＳＳＢ）与修复的初步观察,提示ＤＮＡ损伤也是体外ＴＮＴ性白内障中的早期变化．     

蛋白激酶CK2与DNA断裂修复

蛋白激酶CK2（酪蛋白激酶Ⅱ）是真核细胞中普遍存在的一种信使非依赖的丝氨酸／苏氨酸蛋白激酶,它底物众多,功能广泛。DNA断裂修复是一个涉及很多种酶和蛋白的过程,CK2在其中起着很重要的作用。     

AT细胞DNA双链断裂重接修复效率及其忠实性

用脉冲电场凝胶电泳和双标记基因质粒ＤＮＡ转染技术研究辐射敏感的毛细血管扩张性共济失调症患者皮肤成纤维细胞（ＡＴ５ＢＩＶＡ）和正常辐射抗性的人宫颈癌细胞（ＨｅＬａＳ３）ＤＮＡ双链断裂重接修复率及其忠实性。结果表明γ射线照射诱发ＤＮＡ双链断裂的产额和重接修复率,在两株细胞间无差别．而ＡＴ细胞对导入的限制性内切酶ＥｃｏＲＶ产生双链断裂质粒ＤＮＡ的重接修复忠实性显著低于ＨｅｌａＳ３ｔｅ胞,表明ＡＴ细胞易发生ＤＮＡ错误修复,这很可能就是ＡＴ细胞高度辐射敏感性的主要原因。     

碱性凝胶电泳定量测定非标记DNA的单链断裂

DNA单链断裂(Single Strand Breaks,SSB)的测定是研究DNA损伤与修复的重要检测方法之一。用碱性琼脂糖凝胶电泳测定SSB的最早报告见于1979年,但直到1986年才由Freeman推导出相应的定量计算公式,此后该方法被广泛应用。该法除不需同位素标记和操作简单快捷外,还能够进行定     

γ辐射诱导质粒DNA单链断裂的反向氧效应

以凝胶电泳法研究了γ辐射引起质粒ＤＮＡ单链断裂（ＳＳＢ）的氧效应,发现含自由基清除剂－甘露醇的ＤＮＡ溶液在Ｎ２饱和条件下的ＳＳＢ产额大于空气饱和时的产额,使得Ｓ析氧增比（ＯＥＲ）小于１,即ＤＮＡ之ＳＳＢ存在反向氧效应,同时,Ｇ（ＳＳＢ）及其氧增比均随甘露醇浓度的增大而减小。     

大白鼠肝实质细胞的分离及其DNA单链区与细胞年龄的关系

本文报告了一个分离同一个体中老年、青年肝实质细胞(Parenchy-mal cell)的方法,并利用S_1酶作探针研究了不同年龄肝实质细胞DNA的单链区多寡与细胞年龄的关系。 用胶原酶溶液灌注活体大白鼠肝脏得到游离肝实质细胞。用Percoll作密度梯度介质对同一个体大白鼠的肝实质细胞进行梯度离心分离,得到比重不同的两部分肝实质细胞。再用S_1酶分别降解不同比重肝实质细胞DNA中的单链区域。结果表明:比重较大的肝实质细胞DNA中所含的单链区较比重小的肝实质细胞DNA中的单链区为多。提示这种差异可能与肝实质细胞的年龄有关。用不同年龄大鼠的肝细胞作对比,也证明老年大白鼠肝实质细胞DNA的单链区较多。     

梨形四膜虫衰老过程中DNA含量的变化

应用一种新型的细胞核内DNA含量测定方法──图像分析法,测定真核细胞梨形四膜虫衰老过程中DNA含量的变化.根据Beer-Lambert定律,以细胞核在不同生长期内的积分光密度的水平表示核内DNA含量的变化.该方法具有测量速度快,重复性好,操作简单,结果可靠等优点.实验结果表明:四膜虫在进入对数生长期时,DNA含量逐渐达到高峰,随着细胞逐渐老化,细胞分裂次数及核内DNA含量逐步减少.     

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.     

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.     

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.     

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）。     

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.     

DNA单链二级结构预测与单链构象多态性分析的一致性研究

单链构象多态性(SSCP)分析是一种简便,快速检测DNA突变的方法,它在基因突变检测、遗传分析、进化研究等领域有着广泛的应用价值.但是这种方法的突变检出率随DNA序列不同而变化,一般只能达到70%～80%.这主要是有的碱基突变对单链DNA的构象影响较小,不能通过SSCP检测出来.将计算机对DNA二级结构的预测结果和实验结果作了对比,发现二者有很高的一致性.这一结果表明计算机的DNA单链二级结构预测分析可用于PCR-SSCP分析的辅助设计,提高SSCP的突变检出率.     

电离辐射诱导DNA链断裂的动力学研究

通过测试γ射线辐照下超螺旋ｐＢＲ３２２ ＤＮＡ分子单链断裂（ＳＳＢ）,双链剂量效应,得到ＳＳＢ、αＤＳＢ产额与ＤＮＡ现含一定浓度甘露醇的ＤＮＡ溶液体系中,Ｇ（ＳＳＢ）、Ｇ（αＤＳＢ）的例数与ｃ（ＤＮＡ）的倒数成线性关系,并以二级动力学描述了ＤＮＡ和甘露醇分子对．ＯＨ的竞争反应,得到．ＯＨ引起ＳＳαＤＳＢ的速率常数及其效率。     

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。