不同鼠龄小鼠脾细胞对过氧化氢诱导DNA损伤的修复能力

目的观察青年鼠和老年鼠脾细胞对过氧化氢(H2O2)诱导DNA损伤的修复能力的差异。方法通过S1核酸酶消化实验,对比分析两组小鼠脾细胞在H2O2刺激前后单链DNA的比例及修复程度。通过非程序性DNA合成(UDS)实验观察脾细胞在H2O2刺激后的切除修复水平。结果两组小鼠脾细胞的单链DNA含量与处理前相比均有明显提高,但修复后,青年鼠单链DNA含量的下降比例(13.4%)显著大于老年鼠(6.8%)(P<0.05)。UDS实验表明,两组小鼠均在DNA损伤大约12 h后达到修复高峰,但老年鼠的绝对修复水平大约只有青年鼠的1/3(P<0.01)。结论青年鼠和老年鼠脾细胞对H2O2刺激具有类似的敏感性,但DNA的修复能力随小鼠的增龄显著降低。H2O2通过加速DNA损伤的积累从而促进细胞衰老。     

两种不同方法建立乳鼠神经细胞缺氧/复氧损伤模型

目的研究利用过氧化氢(H2O2)和氯化钴(CoCl_2)建立大鼠乳鼠神经细胞缺氧损伤模型,寻找最适宜的浓度和时间。方法提取原代乳鼠神经细胞,待细胞生长良好后加入不同浓度的H_2O_2和COCl_2作用不同时间,利用MTT法检测细胞存活率。结果 H_2O_2可快速导致细胞缺氧损伤,以600、800μmol/L作用于神经细胞3 h为适宜。CoCl_2引起的细胞损伤较缓慢,以300、400μmol/L作用24 h效果较好。结论 H_2O_2和CoCl_2均可导致细胞氧化损伤,效果确切。     

H_2O_2致小鼠淋巴细胞DNA的损伤及修复

目的　应用单细胞电泳检测 (SCGE)过氧化氢 (H2 O2 )致小鼠淋巴细胞 DNA损伤及其修复。方法　标准 SCGE条件下荧光显微镜观察。结果　 H2 O2 不同浓度 80、16 0和 32 0μmol/ L ,3个组 H2 O2 浓度均可致小鼠淋巴细胞 DNA损伤 ,较低剂量即可造成细胞拖尾 ,可逆性损伤在 6 0 m in内可获得明显修复。结论　单细胞电泳可快速灵敏地检测氧化损伤 ,也可能用于流行病学研究。     

脱氧雪腐镰刀菌烯醇致Vero细胞核基因组DNA损伤与修复的彗星实验观察

目的　应用彗星实验技术观察脱氧雪腐镰刀菌烯醇 (DON)对Vero细胞DNA损伤与修复特征 ,探讨DON的遗传毒性。方法　① 1μmol、5 μmol、10 μmolDON对指数生长期的Vero细胞分别染毒 4、16h后 ,观察DNA的损伤。② 10μmolDON处理的细胞重新孵育 15、3 0、60、12 0min ,观察DNA损伤修复。③以彗星实验技术结合“彗星图像分析软件(KIAS)”获得彗星细胞主要参数 ,并进行统计分析。结果　①随染毒剂量增加 ,染毒时间延长 ,受损伤细胞的数量逐渐增加 ,细胞DNA损伤程度逐渐加重 ;短时间染毒 ( 4h) ,损伤以DNA碎片的数量增加为主 ;较长时间染毒 ( 16h) ,损伤以DNA碎片变小为主。②去除DON ,重新孵育若干时间 ,随修复时间延长 ,受损伤细胞数量逐渐减少 ,细胞DNA损伤程度逐渐减轻 ,提示受损细胞在进行自我修复 ;短时间染毒 ( 4h)修复启动时间较短 ( <15min) ,而长时间染毒 ( 16h)修复启动时间也相应延长 ( >15min) ;孵育 12 0min后 ,修复完成。结论　DON毒素可致Vero细胞DNA损伤 ,重新孵育若干时间后损伤可修复。     

老年大鼠脑突触体谷氨酸,γ—氨基丁酸的代谢变化

采用SD雄性青年大白鼠(3月龄)和老年大白鼠(27月龄),分别取其海马、额皮质和颞皮质制备突触体,观察衰老时,突触体高亲和力摄取和释放Glu、GABA的变化。结果表明:①老年大鼠海马、额皮质和颞皮质突触体对~3H—Glu、~3H—GABA的高亲和力摄取较青年鼠均极显著下降;②老年大鼠海马突触体释放Ser、His、Gly、Thr、Met及GABA显著减少,释放Glu无明显改变;③β—丙氨酸抑制青年鼠突触体摄取~3H-GABA,抑老老年鼠突触体摄取~3H-Glu和~3H-GABA。本文结果提示,衰老时大鼠脑突触体两种递质氨基酸Glu和GABA的代谢减退。     

北京健康人群淋巴细胞基因损伤与修复能力的增龄性变化

目的探讨北京健康人群外周血淋巴细胞DNA损伤与修复能力在氧化应激状态下的增龄性变化。方法以过氧化氢(H2O2)处理各年龄组健康人群外周血淋巴细胞后,用单细胞凝胶电泳法(彗星实验)检测淋巴细胞的Olive尾距(OTM值),并分析其与年龄的关系。结果 50、100、200μmol/L H2O2处理各组淋巴细胞,其OTM值均随龄显著升高,其中,50μmol/L组DNA损伤水平与年龄之间呈显著正相关关系。结论氧化应激状态下,北京健康人群外周血淋巴细胞基因损伤水平随增龄显著增加。     

冬凌草甲素诱导人胰腺癌SW1900细胞DNA损伤及对H2AX蛋白表达的影响

目的 研究冬凌草甲素(ORI)诱导胰腺癌SW1900细胞DNA损伤对磷酸化组蛋白(H2AX)表达的影响.方法 彗星实验检测ORI诱导胰腺癌SW1900细胞DNA损伤的程度.Western印迹检测不同浓度ORI作用胰腺癌SW1900细胞后H2AX蛋白的磷酸化.免疫荧光实验检测Phos-S1981 ATM和γ-H2AX焦点.结果 彗星实验结果表明不同浓度的ORI(20,40,80μmol/L)处理SW1900细胞48 h后,可发生DNA损伤,并且随浓度增加,DNA损伤加重.Western印迹检测不同浓度ORI作用胰腺癌SW1900细胞后,H2AX蛋白发生磷酸化,γ-H2AX随着浓度增加表达增大.免疫荧光实验发现Phos-S1981 ATM和γ-H2AX焦点随着浓度增加表达量增加.结论 ORI可以诱导胰腺癌SW1900细胞DNA损伤;H2AX蛋白发生磷酸化,具体DNA损伤信号通路有待进一步研究.     

4-羟基-2-壬烯酸诱导培养的主动脉内皮细胞DNA损伤

研究 4 羟基 2 壬烯酸对体外培养的主动脉内皮细胞作用 ,以探讨动脉粥样硬化的发病机理。采用单细胞凝胶电泳检测DNA损伤 ,对体外培养的主动脉内皮细胞在不同浓度 4 羟基 2 壬烯酸作用下产生的DNA损伤进行检测。结果发现 ,体外培养的主动脉内皮细胞分别用 5 μmol L、10 μmol L和 15 μmol L 4 羟基 2 壬烯酸处理 10h后彗星试验的尾距分别为 32 .8± 1.1、4 4 .3± 1.0和 74 .6± 1.0 ,与未用 4 羟基 2 壬烯酸处理的正常对照组彗星试验的尾距 (6 .0± 0 .7)比较 ,差异有显著性 (P <0 .0 0 1) ,经 1μmol L 4 羟基 2 壬烯酸处理后的主动脉内皮细胞彗星试验的尾距为 11.3± 0 .9,与正常对照组比较 ,两者之间差异无显著性 (P >0 .0 5 )。结果提示 ,4 羟基 2 壬烯酸可导致体外培养的主动脉内皮细胞的DNA损伤 ,并随着 4 羟基 2 壬烯酸的浓度增高DNA损伤加剧。     

缺锌对衰老小鼠抗氧化系统和肝脏DNA损伤修复功能的影响

目的　通过D 半乳糖诱导小鼠衰老模型 ,探讨缺锌对衰老小鼠抗氧化系统和肝脏DNA损伤与修复的影响。　方法　雄性 3月龄小鼠 70只 ,随机分成 5组 :青年组、衰老模型组、衰老缺锌组、衰老配喂组和衰老补锌组。各衰老组按 10 0mg /kg经颈背部皮下给予D 半乳糖注射液 ,青年组给予等剂量的生理盐水 ,连续 30d。衰老缺锌组和补锌组喂饲缺锌饲料 (含锌 1 6 1μg/kg) ,其他组喂饲正常锌饲料 (含锌 5 0 μg/kg) ,最后 2周补锌组喂饲补锌饲料 (10 0 μg/kg)。第 30天处死小鼠 ,取样检测血清锌、肝锌、超氧化物歧化酶、丙二醇、肝脂褐质和DNA损伤情况。　结果　与衰老模型组相比 ,衰老缺锌组血清锌 (0 5 3± 0 1)mg/L、肝锌 (14 5 4± 2 18)mg/L水平下降 ,血清和肝超氧化物歧化酶活性〔(14 2 87± 10 16 )NU/ml和 (180 11± 13 2 2 )NU/ml,P <0 0 5〕降低 ,丙二醇含量升高 ,肝脂褐质含量增高 ;彗星试验显示衰老缺锌组小鼠肝DNA损伤加重 ,彗星细胞尾长 /总长比值显著增加。补锌后上述指标均有改善。　结论　锌可有效的影响衰老的速度和程度 ,缺锌可加速衰老的进程 ,适当补锌有助于延缓衰老。     

H2O2诱导体外培养的皮肤成纤维细胞氧化应激损伤

目的探讨H2O2对体外培养的人皮肤成纤维细胞生物学特性的影响,从细胞水平为抗皮肤衰老药物的研究提供较理想的皮肤衰老模型。方法用不同浓度的H2O2(50～300μmol/L)干预体外培养的正常皮肤成纤维细胞,MTT比色法检测细胞增殖,流式细胞仪检测细胞凋亡,羟脯氨酸比色法检测胶原含量,紫外分光光度计测定细胞中超氧化物歧化酶(SOD)的活性和丙二醛(MDA)的含量。结果 H2O2浓度依赖性地抑制皮肤成纤维细胞增殖,促进细胞凋亡,降低细胞SOD的活性,减少细胞胶原含量,增加MDA含量。结论氧化应激可损伤皮肤成纤维细胞,用H2O2致皮肤成纤维细胞氧化损伤模型可用于抗皮肤衰老药物的研究。     

Single-molecule denaturation mapping of DNA in nanofluidic channels

Here we explore the potential power of denaturation mapping as a single-molecule technique. By partially denaturing YOYO®-1-labeled DNA in nanofluidic channels with a combination of formamide and local heating, we obtain a sequence-dependent “barcode” corresponding to a series of local dips and peaks in the intensity trace along the extended molecule. We demonstrate that this structure arises from the physics of local denaturation: statistical mechanical calculations of sequence-dependent melting probability can predict the barcode to be observed experimentally for a given sequence. Consequently, the technique is sensitive to sequence variation without requiring enzymatic labeling or a restriction step. This technique may serve as the basis for a new mapping technology ideally suited for investigating the long-range structure of entire genomes extracted from single cells.     

慢性乙型肝炎患者12周自发性HBV DNA水平变化分析

目的 分析慢性乙型肝炎患者12周内自发性HBV DNA水平下降情况.方法 回顾性分析2003-2005年未接受抗病毒药物治疗的慢性乙型肝炎患者12周内自发性HBV DNA水平下降情况,并根据患者基线ALT、总胆红素(TBil)水平进行分组,分析基线ALT、TBil对自发性HBVDNA水平下降的影响.两组间计量资料比较采用t检验或Wilcoxon符号秩和检验;多组数据均值比较采用方差分析;组间率的比较采用x2检验.结果 共收集213例慢性乙型肝炎患者,男性174例,女性39例,年龄18～65(33.0±10.0)岁.其中慢性乙型肝炎轻～中度124例,慢性乙型肝炎重度89例;12周时失访19例(8.92%).所有患者HBV DNA基线均值为(6.66±1.03)log10拷贝/ml,12周时为(5.98±1.53)log10拷贝/ml(P<0.01).慢性乙型肝炎重度患者基线时HBVDNA水平均值低于慢性乙型肝炎轻～中度患者,分别为(6.45±0.99)log10拷贝/ml与(6.81±1.04)log10拷贝/ml(P<0.05);但两组12周时HBV DNA水平均值及HBV DNA水平下降值的差异均无统计学意义.12周时HBV DNA≤3 log10拷贝/ml患者的基线ALT及TBil值高于HBVDNA>3 log10拷贝/ml组,但差异均无统计学意义.12周时HBV DNA水平下降值≥2 log10拷贝/ml与<2 log10拷贝/ml两组患者的基线ALT、TBil水平相近(P>0.05).基线ALT水平≤5倍正常值上限(ULN)与>5×ULN两组患者12周时HBV DNA水平均值及HBV DNA水平下降值的差异均无统计学意义;两组患者12周时HBV DNA≤3 log10拷贝/ml、HBV DNA水平下降值≥2 log10拷贝/ml的比例,差异也无统计学意义(P>0.05).基线时ALT≤5 × ULN及TBil≤5×ULN组HBV DNA水平均值高于其他3组(P<0.05),但12周时各组HBV DNA水平均值、HBV DNA下降值比较,差异无统计学意义. 结论 慢性乙型肝炎患者12周内存在一定程度的自发性HBV DNA水平下降,但肝脏炎症损伤程度与患者12周内自发性HBV DNA水平下降程度无明显的相关性.     

p53 binding to nucleosomes within the p21 promoter in vivo leads to nucleosome loss and transcriptional activation

It is well established that p53 contacts DNA in a sequence-dependent manner in order to transactivate its myriad target genes. Yet little is known about how p53 interacts with its binding site/response element (RE) within such genes in vivo in the context of nucleosomal DNA. In this study we demonstrate that both distal (5') and proximal (3') p53 REs within the promoter of the p21 gene in unstressed HCT116 colon carcinoma cells are localized within a region of relatively high nucleosome occupancy. In the absence of cellular stress, p53 is prebound to both p21 REs within nucleosomal DNA in these cells. Treatment of cells with the DNA-damaging drug doxorubicin or the p53 stabilizing agent Nutlin-3, however, is accompanied by p53-dependent subsequent loss of nucleosomes associated with such p53 REs. We show that in vitro p53 can bind to mononucleosomal DNA containing the distal p21 RE, provided the binding site is not close to the diad center of the nucleosome. In line with this, our data indicate that the p53 distal RE within the p21 gene is located close to the end of the nucleosome. Thus, low- and high-resolution mapping of nucleosome boundaries around p53 REs within the p21 promoter have provided insight into the mechanism of p53 binding to its sites in cells and the consequent changes in nucleosome occupancy at such sites.     

Mutational specificity and genetic control of replicative bypass of an abasic site in yeast

Abasic (AP) sites represent one of the most frequently formed lesions in DNA, and they present a strong block to continued synthesis by the replicative DNA polymerases (Pols). Here we determine the mutational specificity and the genetic control of translesion synthesis (TLS) opposite an AP site in yeast by using a double-stranded plasmid system that we have devised in which bidirectional replication proceeds from a replication origin. We find that the rate, the genetic control, and the types and frequencies of nucleotides inserted opposite the AP site are very similar for both the leading and the lagging DNA strands, and that an A is predominantly inserted opposite the AP site, whereas C insertion by Rev1 constitutes a much less frequent event. In striking contrast, in studies that have been reported previously for AP bypass with gapped-duplex and single-stranded plasmids, it has been shown that a C is the predominant nucleotide inserted opposite the AP site. We discuss the implications of our observations for the mechanisms of TLS on the leading versus the lagging DNA strand and suggest that lesion bypass during replication involves the coordination of activities of the replicative Pol with that of the lesion-bypass Pol.     

密码子优化及其在DNA疫苗中应用的研究进展

近年来,DNA疫苗已在寄生虫病、结核病、艾滋病、肿瘤等领域进行了广泛的研究,但其保护作用常常并不理想.不同物种在密码子使用上的偏性导致外源抗原基冈在不同宿主中不能有效表达,这可能是影响DNA疫苗效能的主要凶素之一.已证实密码子优化后能显著增加蛋白的表达量,提高疫苗的保护率.该文就密码子偏性、密码子优化以及其在疟疾、艾滋病等DNA疫苗中的应用研究进展作一介绍.     

The role of cell-free DNA in predicting colorectal cancer prognosis

Introduction: Colorectal cancer is a cancer of the digestive system with poor prognosis. Cell-free DNA has received much attention with its unique predominance, especially in colorectal cancer.

Areas covered: This study has summarized recent advancements and challenges regarding cell-free DNA in predicting CRC prognosis. Furthermore, the authors make predictions on the potential developments concerning cell-free DNA in future prognosis prediction techniques.

Expert commentary: Cell-free DNA has the value of predicting CRC prognosis as an important biomarke. Further clinical trials should be performed to promote translating cell-free DNA into clinical applications.     

The Promotion of Genomic Instability in Human Fibroblasts by Adenovirus 12 Early Region 1B 55K Protein in the Absence of Viral Infection

The adenovirus 12 early region 1B55K (Ad12E1B55K) protein has long been known to cause non-random damage to chromosomes 1 and 17 in human cells. These sites, referred to as Ad12 modification sites, have marked similarities to classic fragile sites. In the present report we have investigated the effects of Ad12E1B55K on the cellular DNA damage response and on DNA replication, considering our increased understanding of the pathways involved. We have compared human skin fibroblasts expressing Ad12E1B55K (55K⁺HSF), but no other viral proteins, with the parental cells. Appreciable chromosomal damage was observed in 55K⁺HSFs compared to parental cells. Similarly, an increased number of micronuclei was observed in 55K⁺HSFs, both in cycling cells and after DNA damage. We compared DNA replication in the two cell populations; 55K⁺HSFs showed increased fork stalling and a decrease in fork speed. When replication stress was introduced with hydroxyurea the percentage of stalled forks and replication speeds were broadly similar, but efficiency of fork restart was significantly reduced in 55K⁺HSFs. After DNA damage, appreciably more foci were formed in 55K⁺HSFs up to 48 h post treatment. In addition, phosphorylation of ATM substrates was greater in Ad12E1B55K-expressing cells following DNA damage. Following DNA damage, 55K⁺HSFs showed an inability to arrest in cell cycle, probably due to the association of Ad12E1B55K with p53. To confirm that Ad12E1B55K was targeting components of the double-strand break repair pathways, co-immunoprecipitation experiments were performed which showed an association of the viral protein with ATM, MRE11, NBS1, DNA-PK, BLM, TOPBP1 and p53, as well as with components of the replisome, MCM3, MCM7, ORC1, DNA polymerase δ, TICRR and cdc45, which may account for some of the observed effects on DNA replication. We conclude that Ad12E1B55K impacts the cellular DNA damage response pathways and the replisome at multiple points through protein–protein interactions, causing genomic instability.     

维持性血液透析患者淋巴细胞DNA氧化损伤的研究

目的通过彗星(comet)实验研究维持性血液透析(MHD)患者淋巴细胞DNA的氧化损伤程度及透析膜对其的影响。方法于2003年5月至2004年10月选择中国医科大学附属第一医院的20例接受MHD治疗的慢性肾功能不全(CRF)患者,随机分为血仿膜(HE)组和聚砜膜(PS)组。分别使用HE和PS透析12周后采集静脉血,分离淋巴细胞进行comet实验。测量彗星尾长,作为DNA氧化损伤程度的观察指标。同时设立正常对照组。结果MHD患者的彗星尾长显著长于正常对照组(P<0.05),HE组的彗星尾长显著长于PS组(P<0.05)。MHD患者的彗星尾长与血清肌酐(r=0.663,P<0.05)、尿素氮(r=0.612,P<0.05)量呈显著正相关。结论MHD患者体内DNA氧化损伤的增强与血清肌酐、尿素氮量呈正相关;从DNA氧化损伤的角度来评价透析膜的生物相容性,聚砜膜优于血仿膜。