彗星实验检测紫外线诱导的K562细胞DNA损伤

背景与目的研究紫外线(Ultraviolet,UV)诱导K562细胞DNA损伤情况,评价彗星实验改良方法及分析参数的可靠性。材料与方法采用0.3mW/cm2UV以用不同时间(3、5、10、40、60、120、180、240s)照射K562细胞,诱导细胞DNA损伤,用彗星实验检测,CASP软件分析。结果尾长、彗星长、尾矩、Olive尾矩4个参数与紫外线照射时间有良好的时间依赖关系。结论0.3mW/cm2的紫外线照射K562细胞3s即可造成细胞DNA损伤,CASP分析软件可以用于彗星检测分析,其中尾长、彗星长、尾矩、Olive尾矩可作为彗星实验的分析指标,所改良的彗星实验系统可靠性较强,且基本上解决了实验中常见的脱胶现象,采图更方便。     

过钒酸钠对60Coγ射线照射后BET-2细胞增殖反应的影响研究

目的:观察酪氨酸磷酸酶(PTP)特异性抑制剂过钒酸钠(sodiumpervanadate Per)对60Coγ射线照射后造血细胞增殖反应的影响。方法:BET2细胞经60Coγ射线照射0、1、3、5、7和10Gy后,分为Per处理组和无Per组,用MTT法测定BEL2细胞对EPO的增殖反应性。结果:60Coγ射线照射后,BET2细胞对EPO增殖反应性显著降低,并与照射剂量相关;适当浓度Per(1～31.25mmol/L)能促进BET2细胞在无EPO状态下经一定剂量60Coγ射线照射后的细胞存活和增殖。结论:60Coγ射线照射后造血细胞的损伤可能伴有造血因子受体信号转导障碍,调控造血细胞受体信号通路中PTP活性对60Coγ射线照射引发的造血细胞损伤有一定的保护作用。     

FADU法检测~(60)C_0γ-射线照射DNA的损伤及修复

DNA是生物细胞遗传、分裂、增殖的主要物质基础。DNA双链断裂被认为是引起细胞死亡及染色体畸变的主要原因之一,因此它的损伤比细胞中任何其它成分的损伤都更为重要。本文采用了FADU法检测了体外培养的CHL细胞在受(60)~C_0γ射线照射后DNA的双链断裂与剂量之间的关系,同时对温育后断裂的双链DNA修复能力也进行了初步探讨,实验结果表明,在0-20 Gg的剂量范围内(剂量率245尺/分),双链DNA的存留率D％及DNA相对损伤量Q_D与剂量之间存在着明确的剂量效应关系、温育1小时     

宫颈癌放疗患者外周血淋巴细胞DNA损伤的研究

目的：评价宫颈癌放疗患者外周血淋巴细胞的DNA损伤。方法：取15例宫颈鳞癌放疗患者放疗前及放疗累积剂量为10、20、30、40、50、60Gy时的外周静脉血,以彗星实验检测淋巴细胞的DNA损伤。结果：各累积剂量组淋巴细胞DNA拖尾率比照射前显著升高（P〈0.01）,并且在0～60Gy之间,拖尾率与累积照射剂量之间成线性关系,回归方程为y=12.133＋0.230x,相关系数r=0.964（P〈0.05）。各累积剂量组尾长比照射前均增加（P〈0.01）,在照射剂量累积30Gy时,尾长达最大。之后随照射累积剂量的进一步增加,尾长稍有下降,但是与照射前相比仍持续在较高的水平（P〈0.01）,尾长与累积剂量间不成线性关系。结论：宫颈癌患者放疗后可造成外周血淋巴细胞DNA损伤。     

放射治疗对宫颈癌病人DNA损伤及hprt基因突变的初步研究

目的：评价放射治疗对宫颈癌患者造成的遗传学损伤。方法：取15例宫颈鳞癌放疗患者放疗前及放疗累积剂量为0Gy、10Gy、20Gy、30Gy、40Gy、50Gy、60Gy时的外周静脉血,以彗星实验检测淋巴细胞的DNA损伤,采用多核细胞法测hprt基因位点突变率。结果：各累积剂量组淋巴细胞DNA拖尾率比照射前显著升高（P〈0.01）,并且在0-60Gy之间,拖尾率与累积照射剂量之间成线性关系。各累积剂量组尾长比照射前均增加（P〈0.01）,在照射剂量累积30Gy时,尾长均值达最大。尾长与累积剂量间不成线性关系。hprt基因位点突变率在照射后升高,与照射前相比,在照射累积剂量为30Gy、40Gy和50Gy时,显示有统计学意义差别（P〈0.05）。hprt基因突变率与累积剂量间呈现较好的剂量-效应关系。结论：宫颈癌患者放疗后造成外周血淋巴细胞DNA损伤及hprt基因突变,hprt基因突变和彗星实验用于评价放疗造成的损伤,具有较高的敏感性。     

单细胞凝胶电泳技术用于估算大剂量电离辐射的初步探索

目的：初步探索碱性单细胞凝胶电泳（single cell gel electrophoresis,SCGE）方法用于大剂量电离辐射生物剂量估算的可行性。方法：用不同剂量水平（0～16Gy）的60Coγ线照射正常人离体外周血,分别于照射后即刻和1 h后进行SCGE检测,用CASP软件分析血细胞＂彗星＂尾矩（tailmoment,TM）,建立剂量-效应曲线。结果：外周血细胞＂彗星＂尾矩随着照射剂量的增加而增加。以＂彗星＂尾矩为指标,使用统计软件Origin 7.5进行拟合,受照即刻在0～16 Gy剂量范围内彗星TM值与受照剂量D之间的剂量-效应曲线为曲线模式,曲线方程为Y=0.836 7＋0.530 9D＋0.079 9D2（R2=0.996 2,P〈0.05）,而0～8Gy剂量范围内剂量-效应曲线为线性模式,直线方程为Y=0.400 3＋1.129 1D（R2=0.996 3,P〈0.05）。受照1h后0～16Gy剂量范围内,彗星TM值与受照剂量D之间的关系曲线为Y=0.461 6＋0.6560D＋0.062 1D2（R2=0.9984,P〈0.05）,0～8 Gy剂量范围内的关系曲线为Y=0.107 1＋1.128 3D（R2=0.999 5,P〈0.05）。结论：用碱性SCGE分析淋巴细胞＂彗星＂尾矩有望成为一种估算大剂量电离辐射的生物剂量计。     

端粒酶抑制剂叠氮胸苷对HeLa细胞放射性DNA损伤修复的影响

背景与目的:研究端粒酶抑制剂叠氮胸苷(Azidothymidine,AZT)对人宫颈癌HeLa细胞DNA放射性损伤修复的影响,探讨端粒酶在放射诱导的DNA损伤修复中的作用.材料与方法:实验分为空白组,AZT组(400 μmol/L AZT处理HeLa细胞24 h),放射组(2 Gy 60Co γ射线照射),AZT放疗组(400 μmol/L AZT处理HeLa细胞24 h后,用2 Gy 60Co γ射线照射).照射后于0、5、10、30、60、180及360 min分别收集细胞,用端粒重复序列扩增法(PCR-based telomeric repeat amplification protocol,TRAP)-联合酶联免疫吸附法(enzyme linked immunosorbent assay,ELISA)即TRAP-ELISA法检测端粒酶的活性.用单细胞凝胶电泳法检测DNA单链断裂损伤,以彗尾DNA百分含量表示DNA单链断损伤量.结果:HeLa细胞受2 Gy 60Co γ射线照射后10 min,端粒酶活性即开始增加,60 min后增加明显,360 min时达到最高.AZT处理HeLa细胞后,能使端粒酶活性下降约50%,而且能抑制HeLa细胞照射后端粒酶活性的增加(P＜0.05).单细胞凝胶电泳实验表明,2 Gy 60Co γ射线照射HeLa细胞后0～10 min,AZT放疗组与放射组的彗尾DNA百分含量无明显差异(P＞0.05),照后30～360 min AZT放疗组彗尾DNA百分含量均高于放射组(P均＜0.05).结论:AZT能阻抑照射后30～360 min DNA单链断裂的修复,说明端粒酶可能在放射性DNA损伤修复中具有重要作用.     

细胞内活性氧及其 DNA损伤产物在启动细胞癌变中的作用

目的：研究辐射（^60Coγ射线照射）和化学致癌物4-甲基亚硝胺-1-（3-吡啶基）-1-丁酮[4-（methylnirosamino)-1-(3-pyridyl)-1-butanone,NNK]诱发细胞（BEP2D）产生的活性氧（reactive oxygen species,ROS)对DNA氧化损伤及其在启动细胞癌变中的作用。方法：细胞内H2O2和O2^-分别用2′,7′-二氯荧光黄双乙酸盐（DCFH-DA）和氢化乙锭（HE）标记,用流式细胞法测定荧光产物2′,7′-二氯荧光黄（DCF）和溴乙锭（EB）荧光强度;8-羟基脱氧鸟嘌呤（8-hydroxydeoxygunosine,OH8dG)含量用高压液相色谱结合电化学方法（HPLC-ECD）测定;用脂质体包埋法将目标DNA（来自NNK处理和^60Coγ射线照射的BEP2D细胞）转染到受体细胞（C3H10T1/2）中,并用G418筛选阳性克隆细胞;用半固体琼脂试验检测细胞转化能力;用裸鼠成瘤试验鉴定细胞恶性转化。结果：NNK和^60Coγ射线可诱发BEP2D细胞内ROS水平及OH8dG含量显著增高,并显示出良好的剂量效应关系;NNK处理和^60Coγ射线照射的BEP2D细胞DNA分别转染到C3H10T1/2受体细胞后,细胞出现转化特性,裸鼠成瘤试验为阳性。结论：辐射和化学致癌物诱发细胞产生的ROS增高及其对DNA的氧化损伤可能启动了细胞癌变。     

单细胞微凝胶电泳技术的研究和应用

在研究辐射生物效应中, 检测DNA 损伤的方法很多。例如: 细胞遗传学方法染色体畸变、微核、姐妹染色单体互换等等。但上述方法费时费力, 并有一定的局限性。八十年代由O st ing 等首次提出的用微板电泳技术检测单细胞水平DNA 损伤。后经Singh 等进一步改进和完善了单细胞微凝胶电泳Single Cellm icrogel elect ropho resis 简称SCGE) 技术, 又称彗星检测法(Comet assay)。该方法突破了传统局限性, 即设备、操作简便, 所需细胞少、无需放射性示踪剂, 而且灵敏、快速, 整个实验过程从来样到结果分析可在一个工作日完成的新技术。因此, 该技术在国外有关研究领域已得到广泛应用。近年来, 国内一些研究领域也引进和建立此方法。主要用于环境诱突变剂或诱癌剂引起的DNA 损伤ö修复的研究; 癌症病人放、化疗方案的最佳选择及科学预后的研究; 作为生物标记进行生物监测及流行病学、毒理遗传学等诸多领域的研究。目前, 也有一些专家学者将此技术应用于植物细胞的研究。SCGE 技术在本实验室已建立, 其方法是使包埋于琼脂糖中的细胞经裂解去掉细胞浆, 在碱性环境下DNA 双链螺旋成为单链, 在电泳电场作用下DNA 断链离开细胞核向正极迁移出现“彗星”状拖尾现象, 再通过荧光染料溴化乙啶结合DNA 显色检测其损伤程度。为将此技术推广于辐射生物效应体内外研究中, 本实验选用在辐射生物效应中经常用的人血淋巴细胞(HBL ) , 中国仓鼠肺细胞(CHL ) 和兔血淋巴细胞这3 种细胞探索利用此技术检测DNA 损伤的可行性。实验结果表明: SCGE 技术能够对单个细胞DNA 损伤进行研究, 荧光测定核DNA 直径和迁移DNA (即彗星尾) 长度, 而尾长度和尾部荧光强度与DNA 链的断裂呈正相关。由于“彗星”成像是因损伤DNA 在电泳中从核内迁出, 因此它的尾长和密度等是评价与量化电泳结果的重要因素。我们选用上述3 种细胞接受60Co2·线照射后, 经SCGE 技术检测DNA 损伤, 照射组与未照射组DNA 迁移距离经统计学检验, 差异显著性水平A= 0. 5。细胞经照射处理后可引起DNA 电泳迁移, 而迁移长度随照射剂量的增加而延长。此项技术可从受照人员外周血淋巴细胞DNA 电泳迁移长度来估算受照剂量, 从而为评价急性照射和慢性小剂量长期照射的损伤程度提供科学依据。     

敌敌畏、乐果和马拉硫磷诱导HepG2细胞DNA损伤的实验研究

目的：评价敌敌畏（dichlorvos,DDVP）、乐果（dimethoate,DM）和马拉硫磷（malathion,Mal）3种常用有机磷农药单独及两两组合下诱导HepG2细胞DNA损伤效应,并阐明联合作用模式。方法：3种有机磷农药分别单独染毒HepG2细胞4h,联合效应研究采用析因设计分别在高剂量水平和低剂量水平下将3种农药两两混合染毒,同时设溶剂对照（DMSO作用4h）和阳性对照（0.5μmol/ml重铬酸钾染毒2h）,染毒结束后进行彗星试验,采集图像并用CASP软件分析,选择尾部DNA百分比、彗星尾长（tail length,TL）和Olive尾矩（Olive tail moment,OTM）表示DNA损伤强度。结果：3种有机磷农药单独作用时,较高剂量组的尾部DNA百分比、TL和OTM均显著高于对照组（P〈0.01）;析因分析联合效应结果表明,较低剂量水平下,DDVP与DM之间无交互作用（P〉0.05）,DDVP与Mal之间以及DM与Mal之间存在交互作用（P〈0.01）,交互方式均为协同;在较高剂量水平,3种农药两两间均存在交互作用（P〈0.05）,交互方式均为拮抗。结论：敌敌畏、乐果和马拉硫磷单独及两两联合作用均可致DNA损伤,在低剂量和高剂量水平下,存在不同的联合作用模式。     

X线放射治疗对鼻咽癌患者外周血淋巴细胞DNA损伤的研究

背景与目的: 评价X线放射治疗对鼻咽癌患者外周血淋巴细胞DNA的损伤。 材料与方法: 采用碱性单细胞凝胶电泳技术,检测19例鼻咽癌X线放射治疗患者在累积照射剂量为0、2、4、8、10、16 Gy时,外周血淋巴细胞的拖尾率,尾长及Olive尾距。 结果: 鼻咽癌患者接受X线放射治疗后,淋巴细胞拖尾率、尾长在累积照射剂量为2 Gy时即出现明显上升(P<0.01 和 P<0.05),Olive尾矩在累积照射剂量为8 Gy时出现明显上升(P<0.01)。在0～16 Gy照射剂量范围内随着剂量的升高外周血淋巴细胞的拖尾率,尾长及Olive尾距随之增加,且呈剂量-反应关系。 结论: 在本实验条件下,X线放射治疗在低剂量时即对鼻咽癌患者外周血淋巴细胞DNA有损伤作用,其照射剂量与外周血淋巴细胞的拖尾率,尾长及Olive尾距呈剂量-反应关系。     

p53和γ-H2AX作为乙醛引起DNA损伤早期生物标记物的实验研究

目的： 评价乙醛染毒p53野生型人类淋巴母细胞(TK6)后,细胞中DNA损伤标记物p53、γ-H2AX的表达变化,并与彗星试验中的DNA链断裂指标进行敏感性比较,探讨p53和γ-H2AX 作为乙醛引起DNA损伤的早期生物标记物的可能。方法：乙醛在0.5~20 mmol/L的浓度范围分别染毒TK6细胞20 min、2和12 h后,采用高通量的流式细胞术检测肿瘤抑制蛋白total-p53、磷酸化p53(p-p53)以及磷酸化组蛋白(γ-H2AX)的细胞表达率和表达强度(平均荧光强度);同时采用碱性彗星试验检测乙醛引起的DNA链断裂指标(细胞拖尾率、尾长、尾部DNA百分含量以及尾矩)的变化。结果：乙醛染毒TK6细胞12 h后,γ-H2AX的表达强度在15及20 mmol/L浓度下显著升高(P<0.05),p-p53的细胞表达率在0.5~7.5 mmol/L浓度范围内呈现剂量依赖的升高趋势,total-p53的细胞表达率趋势与p-p53相似,但与阴性对照组相比,差异无统计学意义。彗星试验中,细胞拖尾率、尾长、尾部DNA百分含量以及尾矩在5.0~12.5 mmol/L浓度范围内均增加,并呈现剂量依赖性。染毒2 h后,与阴性对照组相比,total-p53和p-p53的细胞表达率在15和20 mmol/L浓度下显著升高(P均<0.05),细胞拖尾率、尾部DNA百分含量以及尾矩在20 mmol/L浓度升高(P均<0.05)。染毒20 min后,3种生物标志物均未见清晰的变化趋势,4种DNA链断裂指标均未见显著变化(P均＞0.05)。结论：乙醛染毒TK6细胞12 h可诱导p-p53细胞表达率升高、γ-H2AX细胞表达强度升高、 total-p53细胞表达率轻微升高,以及细胞拖尾率、尾长、尾部DNA百分含量、尾矩的升高。p-p53比γ-H2AX和DNA链断裂指标在检测乙醛诱导的DNA损伤方面更加敏感。     

彗星试验检测间接诱变剂对小鼠睾丸细胞的DNA损伤

目的：探讨组织原代细胞在检测间接诱变剂中的作用。方法：采用单细胞凝胶电泳试验（彗星试验）对３种典型的间接诱变剂（环磷酰胺、２－氨基芴和二甲基苯蒽）所致的离体小鼠睾丸细胞的ＤＮＡ损伤进行了研究。结果：在一定浓度范围内,３种受试物均可直接引起离体小鼠睾丸细胞ＤＮＡ单链断裂,出现拖尾的彗星细胞,其拖尾细胞百分率和ＤＮＡ迁移长度均随受试物浓度的增加而增加,且有明显的剂量反应关系。结论：原代小鼠睾丸细胞具有一定的代谢活化作用。可以用于彗星试验直接检测间接诱变剂所致的ＤＮＡ损伤研究。     

镉金属硫蛋白对小鼠脾淋巴细胞DNA链损伤和修复的体外实验研究

目的：为研究外源性镉金属硫蛋白对淋巴细胞ＤＮＡ的损伤和修复作用。方法：采用单细胞电泳地（ＳＣＧＥ）检测ＤＮＡ单链断裂（ＳＳＢＳ）,观察在脱金属硫蛋白和不同终浓度ＣｄＭＴ染毒作用３０分钟和６０分钟时ＤＮＡ链损伤,和去除受试物后３０分钟、６０分钟、１２０分钟时ＤＮＡ链损伤的修复。结果：ＤＮＡ－ＳＳＢＳ尾长在Ａｐｏ－ＭＴ作用未中;在中、高剂量ＣｄＭＴ染毒３０和６０分钟时都明显增长。损伤后链修复ＤＮＡ迁移     

Modification of lung cancer susceptibility by green tea extract as measured by the comet assay

Green tea is widely consumed throughout the world and is known to possess various beneficial properties that may affect carcinogen metabolism, free radical scavenging, or formation of DNA adducts. Therefore, it is plausible that green tea extract may modify BPDE-induced DNA damage. In this report, we utilized the comet assay to (1) evaluate BPDE-induced DNA damage as a potential marker of cancer susceptibility and (2) assess the ability of green tea to modify BPDE-induced DNA damage. DNA damage in individual comet cells was quantified by (1) visually measuring the proportion of cells exhibiting migration versus those without and (2) the length of damaged DNA migration (comet tail). We detected a dose-response between BDPE concentration and mean comet tail length in EBV-immortalized lymphoblastiod (lymphoid) cell lines. As the concentration of BPDE increased from 0.5 to 3 microM, the length of the mean comet tail length increased proportionally in the 3590P (derived from a healthy subject) and 3640P (derived from a patient with head and neck cancer) cell lines. In separate experiments using lymphoid cells from 21 lung cancer cases and 12 healthy subjects, the mean comet tail length was significantly higher in the lung cancer cases (80.19 +/- 15.55) versus the healthy subjects (59.94 +/- 14.23) (P < 0.01). Similar findings were observed when analyzing the mean percentage of comet induced cells (84.57 +/- 8.85 and 69.04 +/- 12.50, respectively) (P < 0.01). When green tea extract was added in conjunction with BPDE, there was a notable reduction of the mean comet tail length (13.29 +/- 0.97) as compared to BPDE treatment alone (80.19 +/- 15.55) (P < 0.01) in lung cancer cases. There were no statistical differences between the baseline (no treatments) (12.74 +/- 0.63) and the green tea extract treatment (13.06 +/- 0.97) (P = 0.21). These data suggest the modification of lung cancer susceptibility by the green tea extract. Similar results were observed for the percentage of induced comet cells and the statistical trends were similar for the 12 healthy subjects. This preliminary study demonstrated that the detection of BPDE-induced DNA damage via the comet assay may be a useful biologic marker of lung cancer susceptibility. The differential effects in BPDE-induced DNA damage between lung cancer cases and healthy subjects suggests predisposed cancer susceptibility to lung cancer risk. This reports also demonstrated the chemopreventive effects of green tea extract on BPDE-induced DNA damage. These observations provide further support for the application of the comet assay in molecular epidemiologic studies.     

Gamma-radiation-induced single cell DNA damage as a measure of susceptibility to lung cancer: a preliminary report

The comet assay is a sensitive and rapid method for detecting DNA single-strand and double-strand breaks and the individual cell's DNA repair profile. This pilot study was designed to determine whether the comet assay could measure inherited susceptibility to lung cancer. We applied the comet assay in the alkaline condition to test the DNA damage in gamma-irradiated and untreated cultured peripheral blood lymphocytes of 31 cases with previously untreated lung cancer and 39 controls. For each culture, 200 consecutive cells were examined and the number of cells with DNA uncoiling under electrophoresis () was recorded. The comet tail length (the radius of the nucleus plus the length of the migrated DNA) at 400-fold magnification was measured for the first 50 identified comet cells. The mean number of induced comet cells was significantly higher in cases (96.0+/-45.7) than matched controls (68.9+/-35.8) (P<0.05). However, no significant difference was observed in induced comet tail length between cases and controls. When we categorized the number of comet cells by the 75th percentile value in the controls, a higher number of comet cells was associated with significantly increased risk for lung cancer [odds ratio = 4.8 (confidence intervals of 1.5, 15.2)] after adjustment for age, sex, ethnicity and smoking status. The number of gamma-irradiation-induced comet cells (r=0.499, P<0.05) and comet tail length (r=0.520, P<0.05) correlated with the results on a previously reported lung cancer susceptibility marker, bleomycin sensitivity. Also, the number of gamma-irradiation-induced comet cells correlated with the results of the benzo[alpha]pyrene diol epoxide mutagen sensitivity assay, which quantifies induced chromatid breaks (r=0.275, P<0.05). The comet assay might be a simple and inexpensive tool for detecting genetic susceptibility to lung cancer.     

Risk assessment of renal cell carcinoma using alkaline comet assay

BACKGROUND: DNA damage induced by mutagens has been associated with an individual's susceptibility to cancer. METHODS: In the current study, which involved 193 renal cell carcinoma (RCC) patients and 193 controls, DNA damage before mutagen induction (baseline), after benzo(alpha)pyrene dio epoxide (BPDE) treatment, and after gamma-radiation induction were assayed by comet assay in peripheral blood lymphocytes. Olive tail moments were used as DNA damage parameters. The 5 variables that were analyzed for their associations with RCC risk were baseline, BPDE-induced, gamma-radiation-induced, net BPDE-induced (BPDE-induced subtract baseline), and net gamma-radiation-induced (gamma-radiation-induced subtract baseline) Olive tail moments. RESULTS: Significantly higher Olive tail moments were observed in cases compared with controls at baseline (1.95 vs 1.65; P = .008), after BPDE induction (3.10 vs 2.38; P < .001), and after gamma-radiation induction (4.25 vs 3.47; P < .001). The net BPDE-induced and gamma-radiation-induced DNA damage was also found to be significantly higher in cases compared with controls (P < .001 for both mutagens). Using the 75th percentile Olive tail moments in the controls as the cutoff point, the authors found that high levels of baseline DNA damage, BPDE-induced DNA damage, and gamma-radiation-induced DNA damage were associated with significantly increased risks of RCC, with odds ratios of 1.96 (95% confidence interval [95% CI], 1.26-3.06), 2.70 (95% CI, 1.72-4.23), and 3.13 (95% CI, 1.99-4.92), respectively. Similarly, net BPDE-induced and net gamma-radiation-induced DNA damages were also found to be significantly associated with elevated risks of RCC. CONCLUSIONS: The results of the current study suggest that both baseline and mutagen-induced DNA damages assessed by comet assay are associated with an increased risk of RCC.