狼疮模型鼠体内氧化/抗氧化指标与抗dsDNA抗体水平以及肾脏病变的相关性

目的探讨狼疮样模型小鼠体内的氧化/抗氧化状态,及其与抗dsDNA抗体以及肾脏病变的关系。方法应用分光光度法测定血浆氧化/抗氧化指标,ELISA法测定血浆抗dsDNA抗体浓度。应用两样本t检验,比较狼疮模型鼠与正常对照组间氧化/抗氧化指标以及干预组与非干预组间氧化/抗氧化指标以及自身抗体指标间的差异,采用pearson相关分析比较机体氧化/抗氧化指标与抗体指标间相关性。光镜和电镜下观察肾脏的病理改变,并比较干预组和非干预组间病理改变的差异。结果狼疮模型鼠的丙二醛高于正常对照组(t=4.92,P<0.01),其抗dsNDA抗体水平也高于正常对照组(t=6.53,P<0.01),而总体抗氧化能力水平则低于正常对照组(t=-4.15,P<0.01)。丙二醛和抗dsDNA抗体呈现正相关(r=0.647,P<0.05)总体抗氧化能力与抗dsDNA抗体呈现负相关(r=0.877,P<0.01),总体抗氧化能力与丙二醛呈现负相关(r=0.773,P<0.01)。干预组丙二醛低于非干预组(t=-2.46,P<0.05),组织病理学观察表明,干预组的肾脏病变程度轻于非干预组。结论狼疮模型鼠体内氧化程度较正常对照组高;狼疮模型鼠体内氧化程度与抗dsDNA抗体水平以及肾脏病变之间存在相关性。

国产抗dsDNA抗体检测试剂盒的临床应用

抗双链DNA（dsDNA）抗体是系统性红斑狼疮（SLE）病人血清中重要的标志性抗体之一，对诊断SLE有较高的特异性。本文采用国产酶联免疫吸附法（ELISA）抗dsDNA抗体检测试剂盒，评价其在SLE病人诊治中的临床应用。

dsDNA芯片的DNA结合蛋白质计算机模拟系统

dsDNA芯片(double-stranded DNA microarray)是用于DNA结合蛋白质表达研究的新技术,应用一种新的dsDNA芯片制作方法,研究了dsDNA探针设计,开发了相应的计算机模拟系统——DBP软件。可实现芯片上dsDNA探针的选取以及DNA结合蛋白质检测的实验过程模拟,功能包括模拟酶切、模拟电泳、模拟杂交等实验环节。DBP系统包括了可以不断完善的DNA结合蛋白质数据库。对研究dsDNA 芯片,检测和分析在生物体中DNA结合蛋白质的表达谱,揭示细胞的分化和凋亡、信号转导、DNA转录调控的分子机制有重要意义。

抗双链DNA抗体、抗核小体抗体、抗dsDNA-NcX抗体在系统性红斑狼疮中的研究进展

系统性红斑狼疮（SLE）是一种慢性、复发性、炎症性自身免性疾病，本病好发于育龄期女性，临床表现多样，且多种自身抗体的出现是SLE的重要特征。抗双链DNA抗体（抗ds—DNA抗体）是公认的与SLE病情活动相关的指标，是参与狼疮肾炎发病机制的一种自身抗体，其抗体效价随疾病的活动与缓解而降解，甚至可以转阴。

鲱鱼精dsDNA和联苯胺相互作用的光谱分析

在p H值为7.38的磷酸盐缓冲溶液（phosphate buffer saline,PBS）中,采用荧光光谱和紫外-可见吸收光谱,考察联苯胺（benzidine,BZ）与鲱鱼精双链脱氧核糖核酸（double-stranded deoxyribonucleic acid,ds DNA）的相互作用机制,运用离子强度验证结合体系（BZ-ds DNA）荧光光谱的影响和DNA熔点变化两种方法的结合模式.结果表明,BZ以嵌插方式与鲱鱼精ds DNA结合,结合作用力主要是氢键和范德华力,结合反应为自发过程.实验还发现,当ds DNA质量浓度为8.00×10-6~48.00×10-6g/L时,BZ荧光强度F0和结合体系BZ-ds DNA荧光强度F的比值（F0/F）与ds DNA质量浓度呈线性关系,ds DNA的检测限为2.00×10-6g/L,且其测定不受常见共存金属离子和抗坏血酸等的干扰.因此,基于BZ与ds DNA作用的荧光光谱建立的检测ds DNA的新方法,可用于检测实验室废水中的ds DNA,平均回收率在98.00%~103.00%.

利用dsDNA层层膜检测芬顿试剂对天然DNA的损伤

凭借聚阳离子聚乙烯亚胺(PEI)和带负电荷的DNA存在的相互之间的静电作用交替吸附在玻碳电极表面,进而形成非常有序的{dsDNA/PEI}2层层膜.采用槲皮素作为电活性指示剂来检测芬顿试剂对天然dsDNA的损伤.由于槲皮素在DNA多层膜中具有的非常特别的可逆吸入模式,这中模式能够明显分辨损伤和未损伤DNA多层膜的吸入量.由此检测芬顿反应引起的DNA的氧化性损伤,可以提供一种新的检测DNA损伤的电化学方法.

DNA Double-Strand Breaks,Potential Targets for HBV Integration

Hepatitis B virus(HBV)-induced hepatocellular carcinoma(HCC) is one of the most fre-quently occurring cancers.Hepadnaviral DNA integrations are considered to be essential agents which can promote the process of the hepatocarcinogenesis.More and more researches were designed to find the relationship of the two.In this study,we investigated whether HBV DNA integration occurred at sites of DNA double-strand breaks(DSBs),one of the most detrimental DNA damage.An 18-bp I-SceI homing endonuclease recognition site was introduced into the DNA of HepG2 cell line by stable DNA transfection,then cells were incubated in patients’ serum with high HBV DNA copies and at the same time,DSBs were induced by transient expression of I-SceI after transfection of an I-SceI expression vector.By using nest PCR,the viral DNA was detected at the sites of the break.It appeared that integra-tion occurred between part of HBV x gene and the I-SceI induced breaks.The results suggested that DSBs,as the DNA damages,may serve as potential targets for hepadnaviral DNA insertion and the integrants would lead to widespread host genome changes necessarily.It provided a new site to investi-gate the integration.

Codon evolution in double-stranded organelle DNA: strong regulation of homonucleotides and their analog alternations

In our previous study, complete single DNA strands which were obtained from nuclei, chloroplasts and plant mitochondria obeyed Chargaff’s second parity rule, although those which were obtained from animal mitochondria deviated from the rule. On the other hand, plant mitochondria obeyed another different rule after their classification. Complete single DNA strand sequences obtained from chloroplasts, plant mitochondria, and animal mitochondria, were divided into the coding and non-coding regions. The non-coding region, which was the complementary coding region on the reverse strand, was incorporated as a coding region in the forward strand. When the nucleotide contents of the coding region or non-coding regions were plotted against the composition of the four nucleotides in the complete single DNA strand, it was determined that chloroplast and plant mitochondrial DNA obeyed Chargaff’s second parity rule in both the coding and non-coding regions. However, animal mitochondrial DNA deviated from this rule. In chloroplast and plant mitochondrial DNA, which obey Chargaff’s second parity rule, the lines of regression for G (purine) and C (pyrimidine) intersected with regression lines for A (purine) and T (pyrimidines), respectively, at around 0.250 in all cases. On the other hand, in animal mitochondrial DNA, which deviates from Chargaff’s second parity rule, only regression lines due to the content of homonucleotides or their analogs in the coding or non-coding region against those in the complete single DNA strand intersected at around 0.250 at the horizontal axis. Conversely, the intersection of the two lines of regression (G and A or C and T) against the contents of heteronucleotides or their analogs shifted from 0.25 in both coding and non-coding regions. Nucleotide alternations in chloroplasts and plant mitochondria are strictly regulated, not only by the proportion of homonucleotides and their analogs, but also by the heteronucleotides and their analogs. They are strictly regulated in animal mitochondria only by the content of homonucleotides and their analogs.

Maternal gene Ooep may participate in homologous recombination-mediated DNA double-strand break repair in mouse oocytes

DNA damage in oocytes can cause infertility and birth defects. DNA double-strand breaks （DSBs） are highly deleterious and can substantially impair genome integrity. Homologous recombination （HR）-mediated DNA DSB repair plays dominant roles in safeguarding oocyte quantity and quality. However, little is known regarding the key players of the HR repair pathway in oocytes. Here, we identified oocyte-specific gene Ooep as a novel key component of the HR repair pathway in mouse oocytes. OOEP was required for efficient ataxia telangiectasia mutated （ATM） kinase activation and Rad51 recombinase （RAD51） focal accumulation at DNA DSBs. Ooep null oocytes were defective in DNA DSB repair and prone to apoptosis upon exogenous DNA damage insults. Moreover, Ooep null oocytes exhibited delayed meiotic maturation. Therefore, OOEP played roles in preserving oocyte quantity and quality by maintaining genome stability. Ooep expression decreased with the advance of maternal age, suggesting its involvement in maternal aging.

Double-stranded DNA breaks and gene functions in recombination and meiosis

Meiotic prophase I is a long and complex phase. Homologous recombination is an important process that occurs between homologous chromosomes during meiotic prophase I. Formation of chiasmata, which hold homologous chromosomes together until the metaphase I to anaphase I transition, is critical for proper chromosome segregation. Recent studies have suggested that the SPO 11 proteins have conserved functions in a number of organisms in generating sites of double-stranded DNA breaks （DSBs） that are thought to be the starting points of homologous recombination. Processing of these sites of DSBs requires the function of RecA homologs, such as RAD5 1, DMC 1, and others, as suggested by mutant studies; thus the failure to repair these meiotic DSBs results in abnormal chromosomal alternations, leading to disrupted meiosis. Recent discoveries on the functions of these RecA homologs have improved the understanding of the mechanisms underlying meiotic homologous recombination.

Mechanical properties of double-stranded DNA biofilm with Gaussian distribution

In microcantilever-based label-free biodetection technologies, deflection changes induced by adsorptions of double-stranded DNA （dsDNA） molecules on Au-layer surface are greatly affected by the mechanical, thermal and electrical properties of DNA biofilm. In this paper, the elastic properties of dsDNA biofilm are studied. First, the Parsegian＇s empirical potential based on a mesoscopic liq- uid crystal theory is employed to describe the interaction energy among coarse-grained DNA cylinders. Then, con- sidering a Gaussian distribution of DNA interaxial distance, the thought experiment method is used to derive an analyti- cal expression for Young＇s modulus of DNA biofilm with a stochastic packing pattern for the first time. Results show that Young＇s modulus of DNA biofilm is on the order of 10 MPa. These findings could provide a simple and effective method to evaluate the mechanical properties of soft biofilm on snbstrate.

Adsorption dynamics of double-stranded DNA on a graphene oxide surface with both large unoxidized and oxidized regions

The adsorption dynamics of double-stranded DNA(dsDNA)molecules on a graphene oxide(GO)surface are important for applications of DNA/GO functional structures in biosensors,biomedicine and materials science.In this work,molecular dynamics simulations were used to examine the adsorption of different length dsDNA molecules(from 4 bp to24 bp)on the GO surface.The dsDNA molecules could be adsorbed on the GO surface through the terminal bases and stand on the GO surface.For short dsDNA(4 bp)molecules,the double-helix structure was partially or totally broken and the adsorption dynamics was affected by the structural fluctuation of short dsDNA and the distribution of the oxidized groups on the GO surface.For long dsDNA molecules(from 8 bp to 24 bp)adsorption is stable.By nonlinear fitting of the contact angle between the axis of the dsDNA molecule and the GO surface,we found that a dsDNA molecule adsorbed on a GO surface has the chance of orienting parallel to the GO surface if the length of the dsDNA molecule is longer than 54 bp.We attributed this behavior to the flexibility of dsDNA molecules.With increasing length,the flexibility of dsDNA molecules also increases,and this increasing flexibility gives an adsorbed dsDNA molecule more chance of reaching the GO surface with the free terminal.This work provides a whole picture of adsorption of dsDNA molecules on the GO surface and should be of benefit for the design of DNA/GO based biosensors.

Exposure to Long Magnetic Resonance Imaging Thermometry Does Not Cause Significant DNA Double-Strand Breaks on CF-1 Mice

The purpose of the study was to investigate if the high gradient strength and slew rate used for long MRI-thermometry monitoring could cause DNA double-stranded breaks (DSBs). To this end, an enzyme-linked immunosorbent assay (ELISA) was used to quantify γH2AX, a molecular marker for DSBs, in the blood of mice after a 6-hour exposure to magnetic resonance imaging (MRI). Fourteen CF-1 female mice were separated into 4 experimental groups: Untreated negative control, MRI-treated, MRI-Control, and exposed to ionizing radiation positive control. Untreated negative control was used as a baseline for ELISA to quantify γH2AX. MRI-treated consisted of a 6-hour continuous magnetic resonance imaging (MRI) echo planar imaging (EPI) sequence with a slew rate of 192 mT/m/s constituting a significantly longer imaging time than routine clinical imaging. MRI-control mice were maintained under the same conditions outside the MRI scanner for 6-hours. Mice in the irradiation group served as a positive control of DSBs and were exposed to either 2 Gy, 5 Gy or 10 Gy of ionizing radiation. DSBs in the blood lymphocytes from the treatment groups were analyzed using the γH2AX ELISA and compared. Total protein concentration in lysates was determined for each blood sample and averaged 1 ± 0.35 mg/mL. Irradiated positive controls were used to test radiation dose-dependency of the γH2AX ELISA assay where a linear dependency on radiation exposure was observed (r2 = 0.93) between untreated and irradiated samples. Mean and standard error mean of γH2AX formation were calculated and compared between each treatment group. Repeated measures 1-way ANOVA showed statistically significant differences between the means of irradiated controls and both the MRI-control and MRI-treated groups. There was no statistically significant difference between the MRI-treated samples and the MRI-control groups. Our results show that long MRI exposure at a high slew rate did not cause increased levels of γH2AX when compared to control mice, suggesting that no increase in DSBs was caused by the long MR thermometry imaging session. The novelty of this work contradicts other studies that have suggested MRI may cause DSBs;this work suggests an alternative cause of DNA damage.

肿瘤细胞诱导抗双链DNA自身抗体的产生及其生物学特性

为探讨肿瘤细胞是否能诱导anti dsDNA自身抗体的产生及其特性 ,将灭活的SP2 /0、Wehi 16 4、P815等肿瘤细胞免疫BALB/c小鼠 ,结果均产生了高滴度的anti dsDNA自身抗体。对肿瘤细胞诱导的anti dsDNA自身抗体结合特性的研究表明 ,其结合DNA不具有种属、序列、构象特异性。进一步用cELISA和间接免疫荧光法检测发现 ,anti dsDNA自身抗体还能结合多种肿瘤细胞 。

活性DNA诱导小鼠SLE样综合征的量效关系及其特点

目的:研究活性DNA的剂量与其诱导小鼠SLE样综合征的关系,并对其特点进行全面研究。方法:从ConA活化的小鼠脾淋巴细胞中提取DNA,以不同剂量的活性DNA免疫同系小鼠,用ELISA方法测定IgG类抗dsDNA、抗组蛋白抗体的动态变化以及产生抗体的亚型,用免疫荧光检测抗核抗体核型和肾脏免疫复合物沉积。结果:10μg活性DNA能100％诱导抗dsDNA、抗组蛋白抗体生成,诱导的抗体以IgG1类为主,且小鼠肾脏存在大量免疫复合物沉积。5μg活性DNA诱导小鼠ANA阳性率为25％。结论:10腭活性DNA即可诱导小鼠SLE样综合征,主要诱导自身体液免疫应答。

DNA免疫吸附序贯联合免疫抑制剂治疗系统性红斑狼疮的临床观察

目的:观察DNA免疫吸附治疗对系统性红斑狼疮(SLE)患者抗dsDNA抗体的清除作用以及序贯联合免疫抑制剂治疗的疗效反应。方法:收集2017年8月至2021年8月荆州市中心医院风湿免疫科收治的SLE患者,选择体内抗dsDNA抗体高滴度阳性的活动期SLE患者36例,常规治疗基础上联合DNA免疫吸附治疗,观察患者治疗前、后自身抗体及血液生化指标的变化,吸附治疗后给予免疫抑制剂,随访至少12周。结果:吸附治疗后患者体内抗dsDNA抗体滴度显著下降,平均降幅(66.4%±12.8%),高滴度抗核抗体的患者显著减少。随访中,33例患者病情维持稳定,3例患者在1~2年后,抗dsDNA抗体再次上升,重复行DNA免疫吸附治疗,病情稳定。结论:DNA免疫吸附可以在短期内明显清除抗dsDNA抗体,有助于快速缓解病情,序贯联合免疫抑制剂治疗有助于实现疾病的长期稳定。

不同凋亡水平T细胞来源的ALD-DNA对抗双链DNA抗体产生的影响

研究不同凋亡水平T细胞来源的ALD-DNA(activated lymphocyte-derived DNA)对抗双链DNA抗体产生的影响。用尼龙毛柱法分离小鼠脾脏T细胞,分别提取不同凋亡水平T细胞的DNA定义为未凋亡ALD-DNA、低凋亡水平ALD-DNA、中凋亡水平ALD-DNA和高凋亡水平ALD-DNA,并免疫同系BALB/c小鼠;用ELISA方法检测血清中IgG类抗双链DNA抗体的水平及其亚型;用考马斯亮蓝法检测免疫小鼠尿蛋白的含量。结果显示,ALD-DNA的凋亡水平与其诱导产生的抗双链DNA抗体水平成正相关,二者的相关系数r=0.852;不同凋亡水平的ALD-DNA诱导的抗双链DNA抗体均以IgG1为主,但IgG2a和IgG2b的水平在高凋亡ALD-DNA免疫组有明显增高;低凋亡ALD-DNA、中凋亡ALD-DNA和高凋亡ALD-DNA免疫小鼠均有显著蛋白尿形成,并且高凋亡ALD-DNA免疫组的尿蛋白含量有明显增高。该结果表明,高凋亡水平的ALD-DNA免疫同系小鼠更易诱导高水平的致病性IgG类抗双链DNA抗体产生,凋亡DNA可能在系统性红斑狼疮(SLE)发生发展过程中发挥了重要作用,这为我们能更加深入的理解SLE的发生机制提供了有力的实验依据。

抗SmD1、抗核小体抗体、抗双链DNA抗体在系统性红斑狼疮诊断中的意义

目的 评价血清抗SmD1、抗核小体抗体(AnuA)、抗双链DNA(dsDNA)联合检测在系统性红斑狼疮(SLE)诊断中的价值。方法 40例SLE患者、14例其他结缔组织病患者及20名健康人血清抗dsDNA采用放射免疫法检测,抗SmD1、AnuA用德国IMTEC公司抗核抗体谱线性印迹法检测。结果 SLE患者单项检测抗dsD NA、抗SmD1、AnuA的阳性率分别为42. 5%、80. 0%、72. 5%,三者联合检测后的阳性率为87. 5%,诊断效率为85. 2%。结论 抗SmD1、AnuA、抗dsDNA联合检测提高了诊断SLE的敏感性和诊断效率,可避免因单项检测出现的漏诊情况,对SLE的诊断和治疗有重要意义。

马疫锥虫间接免疫荧光试验测定抗双链DNA抗体和抗核抗体

用马疫锥虫间接免疫荧光试验测305例血清抗双链DNA(dsDNA)抗体和抗核抗体(ANA),其中包括50例系统性红斑性狼疮(SLE),43例其它结缔组织病(CTD),76例银屑病,82例其它疾病和52例正常人对照。结果表明全部23例活动期SLE患者显示抗dsDNA和ANA阳性反应,27例非活动期SLE患者抗dsDNA仅1例阳性而ANA24例(88.9％)阳性。其它各组除1例播散性盘状狼疮和1例肺癌外,抗dsDNA均为阴性反应。

三种不同方法检测抗双链DNA抗体的比较分析

目的比较三种不同方法检测抗双链DNA(ds DNA)抗体的敏感度、特异度和符合度,并探讨其与系统性红斑狼疮(SLE)活动情况的一致性,以指导临床工作。方法采用回顾性分析方法 ,对640例住院患者及健康体检者血清标本进行研究,其中215例SLE患者,用间接免疫荧光法(IIF)、线性印迹法(LIA)、放射免疫分析法(RIA)同时检测抗ds DNA抗体。结果抗ds DNA抗体用IIF、LIA和RIA法在SLE中的检出率分别为40.00%、43.72%、47.44%,与对照组有显著差异,三种方法特异性分别为98.35%、96.24%、94.59%,符合率分别为78.75%、78.59%、78.75%,具有极好的一致性(Kappa>0.8);三种方法在SLE活动期的检出率分别为60.76%、58.23%、60.76%,与SLE稳定期有显著差异,用RIA检测的抗ds DNA抗体的浓度与SLE活动性存在显著的线性趋势(P<0.05)。结论抗ds DNA抗体不仅可以作为SLE患者的诊断标准还对其活动性的评估有着指导作用;三种方法学有着各自的优缺点,有条件的医院建议联合采用两种以上方法相互印证。