应用DNA芯片技术结合核技术对辐射致癌分子机制的研究

DNA芯片技术是新近出现的新的基因分析方法,它是将成千上万个寡核苷酸固定在约厘米大小的硅片上,将待测材料用荧光素或同位素标记,在DNA芯片上与探针杂交,通过激光共聚焦显微镜对芯片进行扫描获取杂交探针的荧光信号,该技术可用于DNA测序,转录情况分析,基因诊断与基因药物设计,突变及多肽性检测遗传作图等方面的研究。     

DNA芯片技术及其在毒理学中的应用

DNA芯片技术是近年来迅速发展起来的分子生物学技术，它将成午上万个寡核苷酸固定在厘米大小的硅片上，将待测的材料用荧光素或同位素标记，在DNA芯片上与探针杂交，通过激光共聚焦显微镜对芯片进行扫描获取杂交探针的荧光信号。该技术在人类基因组计划、DNA测序、转录情况分析、基因诊断等方面得到广泛应用。毒理学是生命科学的重要分支之一，DNA芯片技术在毒学中也有潜在的广泛应用前景。本文就DNA芯片技术的原理，制作方法，信号检测，优点及在毒理学中的应用前景进行综述。     

鼠疫耶尔森菌全基因组DNA芯片的研制及用于比较基因组学分析

目的 研制鼠疫耶尔森菌全基因组DNA芯片,并将其用于比较基因组分析。方法 挑选出4005条鼠疫耶尔森菌基因,PCR扩增各基因,以纯化的PCR产物点样制备芯片,采用双色荧光杂交策略,进行芯片比较基因组杂交。结果 设计了若干质控杂交组合,芯片杂交结果与全基因组测序结果完全一致。结论 成功建立了基于全基因组DNA芯片的鼠疫耶尔森菌比较基因组学技术平台。     

应用肿瘤相关寡核苷酸芯片筛选乳癌差异表达基因

目的：应用寡核苷酸芯片技术研究乳癌基因表达谱。方法：根据文献获取目的基因，查询相应基因mRNA序列，通过计算机设计并合成探针，将探针点样于经化学修饰的载玻片上，制备含288种基因的肿瘤相关基因寡核苷酸芯片。提取乳癌与相应正常乳腺组织总RNA，通过逆转录制备荧光标记的cDNA探针并与芯片杂交，经洗片后扫描获取图像，计算机分析比较乳癌组织与正常乳腺组织的差异表达基因。结果:检测16例乳癌组织与正常乳腺组织标本，有10例基因表达存在明显差异，其中表达增高的有6种，表达降低的有4种。结论：乳癌组织与正常乳腺组织存在部分差异表达的基因，乳癌的发生发展与这些基因密切相关；寡核苷酸芯片技术在乳癌相关基因的研究中具有重要意义。     

应用辣根过氧化物酶化学发光法检测毒素原性大肠杆菌方法的建立

利用活化酶标复合物（HRP－PBQ－PEI＋－NH3＋）直接标记毒素原性大肠杆菌不耐热肠毒素（ETEC－LT）基因片段（0．8kb）作探针。HRP在戊二醛作用下与单链靶DNA形成DNA和酶的共价复合物，与DNA探针结合的辣根过氧化物酶（HRP）催化相应的发光刺，经增强型化学发光反应（ECL）在普通X光胶片上自显影（CPD）检测ETEC－LT。结果可检测到0．03pg的纯质粒DNA和103的ETEC菌细胞，dotblot杂交证明该酶标基因探针仅与产LT肠毒素的ETEC杂交，其余均未见杂交阳性反应。结果表明酶（HRP）标基因探针化学发光法检测ETEC是一种简便、快速、安全，高度敏感和特异的非放射性标记基因探针检测技术。     

应用寡核苷酸芯片技术检测IGF-Ⅱ基因SNP方法的建立及其在运动能力相关基因研究中的应用

目的 :杰出运动能力受控于基因与环境的相互作用 ,由多基因控制。寡核苷酸芯片技术是近年出现的一类基因结构分析技术 ,此项技术出现使基因突变检测更加程序化和规模化。本研究选取与运动能力有关的胰岛素生长因子Ⅱ (IGF -Ⅱ )基因为该方法学及其应用研究的突破点 ,尝试把DNA芯片技术引入体育科研。方法 :寡核苷酸芯片技术。结果 :(1)IGF -Ⅱ基因SNP的分析中 ,基因组DNA采用 1∶2 0的套式扩增 ,杂交液中加氯化四甲氨 ,杂交温度 4 2℃ ,杂交时间 6 0min效果最好 ;(2 )国家自行车队的 6名队员基因型相同。结果表明 :(1)PCR产物可以直接用于检测突变 ,不对称PCR产物杂交阳性点信号更强 ;(2 )由碱基组成计算的Tm值与其实际的Tm值有一定的差距 ,会影响杂交温度 ;(3)有效的DNA载波片的处理效果直接影响杂交结果 ,是芯片制备中关键环节之一 ;(4)IGF -Ⅱ是研究运动能力相关基因的一个很好的候选基因。     

基于随机聚合酶链反应的病原菌基因芯片检测技术

目的建立一种基于随机聚合酶链反应的病原细菌基因芯片筛查检测技术。方法用生物学软件分析7种病原菌特异性基因序列的保守性区域,利用Oligo 6.0软件设计针对靶细菌的一系列探针,制备检测用基因芯片。细菌基因组DNA在随机引物扩增中掺入氨基丙烯-dUTP,产物偶联荧光染料后与芯片上探针杂交,通过芯片扫描仪和图像分析软件对结果进行判断。选取19种病原菌基因组DNA进行芯片特异性验证和灵敏度评价,使用问号钩端螺旋体对应靶探针进行基因芯片检测方法重复性验证,并制备问号钩端螺旋体模拟水污染样本进行芯片检测。结果在均一的杂交条件下4种靶细菌均能得到相应特异性杂交图谱,其他非目的细菌均为阴性结果,3种靶细菌基因组DNA最低检测浓度为14.43～363.4 pg/μl,芯片重复性变异系数CV值〈15%,最低可检测含问号钩端螺旋体7×105条/ml模拟水污染样本。结论初步建立的随机聚合酶链反应结合芯片技术的检测方法可用于多种病原菌筛查检测,为细菌高通量筛查与鉴定技术提供了新的思路和实验依据。     

基于随机聚合酶链反应的病原菌基因芯片检测技术

目的建立一种基于随机聚合酶链反应的病原细菌基因芯片筛查检测技术。方法用生物学软件分析7种病原菌特异性基因序列的保守性区域,利用Oligo 6.0软件设计针对靶细菌的一系列探针,制备检测用基因芯片。细菌基因组DNA在随机引物扩增中掺入氨基丙烯-dUTP,产物偶联荧光染料后与芯片上探针杂交,通过芯片扫描仪和图像分析软件对结果进行判断。选取19种病原菌基因组DNA进行芯片特异性验证和灵敏度评价,使用问号钩端螺旋体对应靶探针进行基因芯片检测方法重复性验证,并制备问号钩端螺旋体模拟水污染样本进行芯片检测。结果在均一的杂交条件下4种靶细菌均能得到相应特异性杂交图谱,其他非目的细菌均为阴性结果,3种靶细菌基因组DNA最低检测浓度为14.43～363.4 pg/μl,芯片重复性变异系数CV值<15%,最低可检测含问号钩端螺旋体7×105条/ml模拟水污染样本。结论初步建立的随机聚合酶链反应结合芯片技术的检测方法可用于多种病原菌筛查检测,为细菌高通量筛查与鉴定技术提供了新的思路和实验依据。     

应用辣根过氧化物酶化学发光检测毒素原性大肠杆菌方法的建立

利用活化酶标复合物（HRP－PBQ－PEI^ －NH3^ ）直接标记毒素原性大肠杆菌不耐热肠毒素（ETEC－LT）基因片段（0.8kb)作探讨。HRP在戊二醛作用下与单链靶DNA形成DNA和酶的共价复合物，与DNA探针结合的辣根过氧化物酶（HRP）催化相应的发光剂，经增强型化学发光反应（ECL）在普通X胶片上自显影（CPD）检测ETEC－LT。结果可检测到0.03pg的纯质粒DNA和10^3的ETEC菌细胞，dot blot杂交证明该酶标基因探针仅与产生LT肠毒素的ETEC杂交，其余均未见杂交阳性反应，结果表明酶（HRP）标基因探针化学发光法检测ETEC是一种简便，快速，安全，高度敏感和特异的非放射性标记基因探针检测技术。     

SM耐药结核杆菌rpsL88codon突变发夹探针检测研究

目的：应用发夹DNA探针检测结核杆菌耐链霉素（SM）rpsL88codon点突变,并对照测序结果。方法：运用软件Beacon designer设计rpsL88codon的发夹DNA探针,建立其扩增体系及发夹DNA探针芯片检测方法;比较相应扩增产物测序结果。结果：通过ScanArray Express观测到标准株及耐SM-PCR产物与发夹DNA探针杂交后信号区别明显;耐SM组rpsL88codon突变检出率为60％,发夹DNA探针检测方法与测序法符合率85％。结论：rpsL88codon突变是结核杆菌耐链霉素的主要原因之一,发夹DNA探针技术是一种高灵敏的核酸点突变检测技术,并与测序结果有较好的检测符合率。     

Threshold dose–response in radiation carcinogenesis: an approach from chronic β-irradiation experiments and a review of non-tumour doses

Purpose : To discuss the threshold dose problem in radiation carcinogenesis after a review of the present author's experimental data on mouse tumour induction by chronic β -irradiation and other relevant data. Conclusions : A threshold dose-response in radiation carcinogenesis appears in certain tissues and under certain conditions. The optimum condition for demonstrating an apparent threshold is with partial-body chronic or repeated radiation rather than with acute whole-body radiation. Its possible mechanism is host tolerance, involving DNA repair, apoptosis and an immune response activated by low radiation doses. This tolerance level was examined by a survey in the literature of non-tumour-inducing doses, D nt, the highest dose at which no significant increase of tumours was observed above the control level.     

微卫星DNA及其在放射医学领域中的应用前景

微卫星 DNA(MSDNA)是真核生物基因组重复序列中的主要组成部分之一。由于其分布广、多态性高、自身突变率低、易检测等特点 ,脱颖而出 ,成为优秀的遗传标志 ,广泛用于基因定位、基因作图、种群研究、个体识别 ,并与肿瘤、某些遗传疾病密切相关。射线作为一种环境致突变因子 ,可引起遗传改变及肿瘤形成 ,因此 ,MSDNA近年来在放射医学领域也有较为广泛的应用。     

电离辐射诱导的DNA甲基化模式改变及其在辐射致癌机制研究中的意义

越来越多的研究已经表明,表观遗传机制在癌症发生发展中扮演了非常重要的角色,但是对于辐射致癌的表观遗传机制的研究仅刚刚起步。本文介绍了表观遗传和一种重要的表观遗传标志——DNA甲基化的相关概念,并综述了近年来人们对电离辐射诱导的DNA甲基化模式改变的研究以及DNA甲基化模式改变在辐射致癌机制研究中的意义。     

Radiation-induced genomic instability: are epigenetic mechanisms the missing link?

PURPOSE: This review examines the evidence for the hypothesis that epigenetics are involved in the initiation and perpetuation of radiation-induced genomic instability (RIGI). CONCLUSION: In addition to the extensively studied targeted effects of radiation, it is now apparent that non-targeted delayed effects such as RIGI are also important post-irradiation outcomes. In RIGI, unirradiated progeny cells display phenotypic changes at delayed times after radiation of the parental cell. RIGI is thought to be important in the process of carcinogenesis; however, the mechanism by which this occurs remains to be elucidated. In the genomically unstable clones developed by Morgan and colleagues, radiation-induced mutations, double-strand breaks, or changes in messenger RNA (mRNA) levels alone could not account for the initiation or perpetuation of RIGI. Since changes in the DNA sequence could not fully explain the mechanism of RIGI, inherited epigenetic changes may be involved. Epigenetics are known to play an important role in many cellular processes and epigenetic aberrations can lead to carcinogenesis. Recent studies in the field of radiation biology suggest that the changes in methylation patterns may be involved in RIGI. Together these clues have led us to hypothesise that epigenetics may be the missing link in understanding the mechanism behind RIGI.     

Threshold dose-response in radiation carcinogenesis: an approach from chronic beta-irradiation experiments and a review of non-tumour doses

PURPOSE: To discuss the threshold dose problem in radiation carcinogenesis after a review of the present author's experimental data on mouse tumour induction by chronic beta-irradiation and other relevant data. CONCLUSIONS: A threshold dose-response in radiation carcinogenesis appears in certain tissues and under certain conditions. The optimum condition for demonstrating an apparent threshold is with partial-body chronic or repeated radiation rather than with acute whole-body radiation. Its possible mechanism is host tolerance, involving DNA repair, apoptosis and an immune response activated by low radiation doses. This tolerance level was examined by a survey in the literature of non-tumour-inducing doses, D(nt), the highest dose at which no significant increase of tumours was observed above the control level.     

Introduction

Abstract Purpose: The target cells for radiation carcinogenesis are widely held to be stem or stem-like cells. Classically, stem cells are considered to be those capable of renewing tissues while differentiated cells lose the potential to replicate. More recently it has become apparent that greater developmental plasticity exists and that cells can be reprogrammed to form induced pluripotent stem cells. Modelling of radiation cancer-risk requires understanding of the characteristics, numbers and responses of target stem cells to radiation. Therefore progress in understanding mechanisms of radiation-induced carcinogenesis is dependent on knowledge of stem cell radiobiology. Results: In this context, the European Community's network of excellence on low dose radiation risk called, 'Low Dose Research towards Multidisciplinary Integration (DoReMi)' ( www.doremi-noe.net ) and the United Kingdom's Health Protection Agency organised a workshop on Stem Cells and DNA damage in Oxfordshire on 7/8 December 2011 to address issues relating to radiation, DNA damage and stem cells. In keeping with the aim of improving understanding of low dose ionising radiation health risk, a panel of experts in stem cells and radiobiology were invited to this workshop. This summary includes all presentations at this workshop and is accompanied by full reports of several speakers.     

线粒体作为放射损伤防治靶点的研究进展

电离辐射会导致蛋白质、DNA等生物大分子损伤,以致细胞癌变、凋亡、衰老等一系列变化。放射损伤后,有效防治靶点的缺乏又致可用治疗药物种类十分有限。新近研究提示,线粒体在放射损伤中发挥重要作用,并有望成为新的防治靶点。本研究就电离辐射对线粒体的影响,特别是氧化应激的发生,以及线粒体在电离辐射诱导生物损伤效应中的作用进行综述,旨在讨论将线粒体作为潜在的放射损伤防治靶点的可行性。     

INTRODUCTION

Abstract

Purpose: The target cells for radiation carcinogenesis are widely held to be stem or stem-like cells. Classically, stem cells are considered to be those capable of renewing tissues while differentiated cells lose the potential to replicate. More recently it has become apparent that greater developmental plasticity exists and that cells can be reprogrammed to form induced pluripotent stem cells. Modelling of radiation cancer-risk requires understanding of the characteristics, numbers and responses of target stem cells to radiation. Therefore progress in understanding mechanisms of radiation-induced carcinogenesis is dependent on knowledge of stem cell radiobiology.

Results: In this context, the European Community's network of excellence on low dose radiation risk called, ‘Low Dose Research towards Multidisciplinary Integration (DoReMi)’ (www.doremi-noe.net) and the United Kingdom's Health Protection Agency organised a workshop on Stem Cells and DNA damage in Oxfordshire on 7/8 December 2011 to address issues relating to radiation, DNA damage and stem cells. In keeping with the aim of improving understanding of low dose ionising radiation health risk, a panel of experts in stem cells and radiobiology were invited to this workshop. This summary includes all presentations at this workshop and is accompanied by full reports of several speakers.     

Gamma Radiation Response and Recovery Studies in Radiation Sensitive Mutants of Diploid Yeast

Summary

Cell killing and other deleterious biological effects of ionizing radiation are the result of chemical changes to critical targets, initiated at the time of exposure. Electron-affinic radiosensitizers act, primarily, by chemically modifying this radiation damage and its consequent biological expression, and such changes can be used to probe the nature of the cellular radiation target. According to a redox hypothesis of radiation modification, the molecular mechanism of electronic-affinic radiosensitization involves an oxidative interaction of the sensitizer with reactive, potentially damaging target radicals, which competes with reductive processes that restore the target to its undamaged state.

The effects have been compared of a series of hypoxic cell radiosensitizers on radiation-induced DNA damage and mammalian cell killing, in order to ascertain the nature of the critical radiation target site(s) involved. Sensitizer efficacy is determined by the ability to oxidize the radiation target and is found to increase exponentially with increasing electron affinity. The threshold redox potential, below which no sensitization occurs, corresponds to the oxidation potential of the target bioradical involved, and is characteristic, and useful in identification, of the particular radiation target.

Model product analysis studies of DNA base damage, inorganic phosphate release, single-strand breaks and incorporation of radioactively labelled sensitizer into DNA show a correspondence between the electronic-affinic radiosensitization of DNA damage and cell killing. A careful comparison of the radiosensitization of different DNA sites and cell killing indicates that the sugar-phosphate backbone of DNA, not the heterocyclic bases, is the DNA target site which mimics cell killing in its threshold redox potential and overall radiosensitization response. These results suggest that the enhancement by electron-affinic drugs of radiation damage to the DNA backbone (strand breaks) correlates strongly with, and is the most likely cause of, the radiosensitization of hypoxic cell killing.