线粒体DNA损伤和自由基

线粒体DNA (mitochondricalDNA ,mtDNA)是唯一存在细胞核以外的遗传物质。自由基是造成mtDNA损伤的主要原因 ,以线粒体DNA点突变和缺失突变多见。mtDNA损伤可以导致线粒体氧化磷酸化功能障碍、细胞凋亡和死亡 ,引起对能量需求较高的中枢神经系统、肌肉等组织出现功能障碍甚至疾病。     

精子DNA损伤与保护

精子DNA损伤是引起不育的重要原因,携有DNA损伤的幸存精子可能逃避体内精子选择机制而成熟并将遗传缺陷传递于后代。因此对精子DNA损伤的研究已成为生殖医学的热点之一。损伤精子DNA的因素主要包括氧化应激、微量元素、精子毒性物质、放射线等,而机体则凭借高度压缩精子DNA、抗氧化系统等机制保护精子DNA的完整性。此外,一些药物如抗氧化剂、黑茶提取物等也可以促使这种保护机制的健全与重建。     

精子DNA完整性与精液参数的相关性研究

目的 探讨精子DNA完整性与精液参数之间的相关性.方法 收集2009年3月至2009年7月在解放军105医院生殖中心就诊的220例男性患者的精液标本,采用吖啶橙荧光染色法(AOT)检测精子核DNA碎片指数(DFI).按DFI值分为DFI≤30%、30%50%3组,对DFI与各项精液参数的相关性进行分析.结果 精子活率、活力力、正常形态率随DFI值的升高逐渐降低,各组间均有显著性差异(P<0.05);DFI与精子活率、活动力、正常形态率均呈显著负相关(P<0.05),与精液量及密度无显著相关性.结论 精子DNA损伤可导致精子形态学异常和活力下降,精子DFI是评估男性生育力的一个较好的参考指标.     

精子DNA完整性与精液常规指标相关性分析

目的 探讨精子DNA完整性与精液常规指标的关系.方法 按照WHO<人类精液及精子-宫颈粘液相互作用实验室检验手册>要求对370例男性患者进行精液常规检测及精子DNA完整性分析.以上述手册设定的各精液常规检测指标参考值为依据分组,比较各组间精子DNA完整性是否存在差异,进而分析精子DNA完整性与各精液常规指标的相关性.结果 370例患者中,以不同年龄(岁)(≤30和＞30)、精子活率(%)(≥60和＜60)、前向运动精子活力(%)(≥50和＜50)分组,比较各组间精子DNA完整性无统计学差异(P＞0.05);而以不同精子密度(106/ml)(＜20和≥20＝、精液粘稠度(适中和粘稠)分组,各组间精子DNA完整性比较存在统计学差异(P＜0.05),其中不同密度精子DNA完整性差异显著.结论 与精子数量相关的精子发生过程是影响精子DNA完整性的主要原因;年龄因素及精子运动能力相关的附性腺功能与精子DNA完整性无关;选择合适的精液处理方式,尽量不破坏精子DNA完整性对于男科实验室精液检测及辅助生育技术过程中受精所需的精子悬液的制备等至关重要.     

DNA甲基转移酶的作用及甲基化在肿瘤发生中的作用

表遗传学(epigenetics)调节与肿瘤相关基因的转录调控密切相关.DNA甲基化就是基因表遗传学调节的重要机制之一.DNA异常甲基化与肿瘤的发生和演进关系密切,而DNA甲基化是由DNA甲基转移酶(DNA methyltransferase,DNMT)催化发生并维持的.因此,深入研究DNA甲基转移酶的作用对认识异常甲基化的发生以及异常甲基化在肿瘤的发生和演变中的作用有着重要意义.     

Studies on the Distribution of Feulgen-DNA Content in Normal and Pathological Human Spermatozoa

Untersuchungen über die Verteilung des Feulgen-DNA-Gehaltes in normalen und pathologischen menschlichen Spermatozoen
Die Entwicklung eines Scan-Tisches mit einer Schrittgröße von 0.25 μm erlaubte es uns, genauere Untersuchungen über die Feulgen-DNS-Verteilung in normalen und pathologischen menschlichen Spermatozoen durchzuführen.
Die Art der Verteilung der Feulgen-DNS ließ keine Unterschiede zwischen normalen und pathologischen Spermatozoen erkennen.
Die Wahl der Fixierung, die Hydrolysedauer und die Passage von Cervixmucus führten jedoch zu signifikanten Unterschieden der Feulgen-DNS-Werte und ihrer Verteilung in normalen und pathologischen Spermatozoen.
Das Verteilungsmuster der Feulgen-DNS vermag wahrscheinlich Informationen über die Sekundär- und Tertiärstruktur der Spermatozoen-DNS zu vermitteln.     

精子DNA损伤的机制及检测方法研究进展

DNA是精子的遗传物质,其完整性易受各种因素影响而受损,继而影响精子质量、最终导致不孕不育.其损伤机制涉及染色质组装异常、氧化应激、凋亡异常等.精子DNA完整性受损的患者,用传统的精液检查往往找不到不育原因,因此,对这部分患者进行精子DNA完整性检查具有更重要的意义.目前已经建立了很多检测方法.现就精子DNA完整性受损的机制、常用检测方法及其临床应用作一综述.     

肝部分切除术后肝细胞DNA合成及细胞周期变化的实验研究

目的 本文探讨大鼠肝切除术后肝细胞再生时DNA的合成、细胞的增殖周期及增殖系数的变化。方法 采用大鼠部分肝切除模型．体外培养肝细胞，观察术后一周内大鼠肝细胞DNA合成和细胞周期的变化。结果 术后残肝逐渐长大，一周时达到高峰，但在术后48小时内，增长速度较慢。肝细胞DNA合成和增殖系数在后术24小时内增长较快，且在24小时点达到高峰，以后缓慢下降。结论 肝部分切除术后肝细胞的再生可分为三个时期．这种分期观点对如何防止术后肝衰及降低死亡率具有重要意义。     

Comparison of Colprimetric and Fluorometric Methods for Estimation of Sperm Concentration in Human Ejaculate and Rat Epididymal Sperm

Estimation of sperm concentration with ethidium bromide was found to be simple and rapid. The estimated value of DNA per human spermatozoon, 2.7 pg, agreed with published values. The method is also applicable to rat epididymal spermatozoa, although the apparent concentration of DNA is only 1.5 pg/spermatozoon. In spectrophotometry, falsely high values were noted. These were, however, reproducible and did not interfere with sperm estimation.
Although fluorometry is not more sensitive than spectrephotometry, it is simpler, avoiding corrosive reagents, and more rapid.
A standardized method is outlined and calibration equations, based on correlation analysis, are presented.     

Isolation and characterization of a DNA synthesome from a neuroblastoma cell line

Background

Despite significant analysis of the chromosomal abnormalities associated with neuroblastoma (NB), the role that NB DNA replication may play in the accumulation of genetic damage is poorly understood. For that matter, the mechanisms involved in NB DNA synthesis have yet to be elucidated. In an effort to investigate this process in NB, we have isolated and purified a multiprotein DNA replication complex from human NB cells (IMR-32).

Methods

Using a series of subcellular fractionations, ion-exchange chromatography, and gradient sedimentation steps, we have isolated a simian virus 40 replication competent multiprotein complex from IMR-32 NB cells, which has been designated the DNA synthesome. Enzymatic and immunodetection techniques were used to characterize the multiple components of the multiprotein DNA replication complex.

Results

The NB DNA synthesome was found to remain intact and functional through all the steps of its purification. The proteins and enzymatic activities that were found to copurify with the NB DNA synthesome include: DNA polymerases α, δ, and ?, proliferating cell nuclear antigen, replication factor A, replication factor C, topoisomerases I and II, flap endonuclease 1, and DNA ligase I.

Conclusion

Although the cooperative integration of a DNA replication macromolecular complex (DNA synthesome) is not new, we extend the view of the DNA synthesome mediating DNA synthesis for human NB. The data reported here characterize the human NB DNA synthesome for the first time and provide the groundwork for investigating whether the NB DNA synthesome contributes to faulty DNA replication and tumor pathogenesis for this childhood malignancy.     

Loss and apoptosis of smooth muscle cells in intracranial aneurysms studies with in situ DNA end labeling and antibody against single-stranded DNA

Summary Pathological specimens were collected from 14 unruptured and 13 ruptured aneurysms at the time of clipping and studied in order to assess the underlying mechanism of rupture by investigating degeneration of the aneurysmal wall and possible involvement of apoptosis. Immunohistochemistry with anti-actin antibody showed few smooth muscle cells in the ruptured aneurysms and replacement of the muscularis layer by a fibro-hyalin tissue. However, at least one layer of smooth muscle cells was clearly observed in the unruptured aneurysms. Thus, smooth muscle cells in the wall of the ruptured aneurysms were much more degenerated than those in the wall of unruptured aneurysms. In addition, unruptured aneurysms with an angiographically smooth wall showed well-layered positive staining for anti-smooth muscle actin antibody while those with irregular shapes rarely reacted. We found, for the first time, evidence of DNA fragmentation in the aneurysmal wall. Apoptotic bodies were detected by means of a terminal transferase (TdT)-mediated dUTP biotin nick end labelling technique (TUNEL) and an anti-single-stranded DNA antibody in 54% (7/13) of the ruptured aneurysms. In contrast, apoptotic bodies were found in only 7% (1/14) of the unruptured cases. These results suggest that apoptotic cell death might be involved in the rupture of aneurysms.     

硫喷妥钠对CpG DNA诱导人外周血单个核细胞NF-κB表达和TNF-α、IL-6释放的影响

目的 探讨硫喷妥钠对CpG DNA诱导人外周血单个核细胞(hPBMC)核因子-κB(NF-κB)表达及肿瘤坏死因子α(TNF-α)、白细胞介素-6(IL-6)释放的影响.方法 采集健康志愿者外周血,使用淋巴细胞分离液(Ficoll-Hypaque)密度梯度离心分离hPBMC.分别加入不同浓度的硫喷妥钠后(0.2～1.0 mg/ml),ELISA测定hPBMC培养上清中TNF-α和IL-6浓度,确定硫喷妥钠对CpG DNA刺激细胞分泌TNF-α及IL-6的抑制作用及其剂量-效应关系和时间-效应关系;免疫蛋白印迹法(Western blotting)分析hPBMC胞核NF-κB表达.结果 不同浓度的硫喷妥钠(0.2～1.0mg/ml)显著抑制CpG DNA诱导hPBMC NF-κB P65表达和TNF-α、IL-6释放(P<0.05或P<0.01).提前1～4 h给予硫喷妥钠或同时给予硫喷妥钠和CpG DNA(0 h),其拮抗CpG DNA诱导细胞因子释放的作用非常显著(P<0.01),且硫喷妥钠在刺激物CpG DNA给予后1 h再加入,也能观察到显著拮抗作用(P<0.05).结论 硫喷妥钠可通过下调CpG DNA诱导hPBMC NF-κB P65表达,从而降低促炎细胞因子TNF-α和IL-6的释放.     

一个多发性骨骺发育不良大家系的临床特点及致病基因分析

目的通过对一个5代疑似多发性骨骺发育不良(multiple epiphyseal dysplasia,MED)的大家系(患者17例)进行临床特征分析和致病基因的筛查,为遗传咨询和产前分子诊断提供实验依据。方法采集家系成员病史,一般体检、关节、髋部X线片资料;收集该家系外周血样,提取样本DNA,靶向基因高通量测序方法对先证者DNA临床全外显子进行测序,使用Next Gene软件对测序序列进行比对分析,并进一步利用Ingenuity软件对存在的突变进行功能注释,寻找先证者致病突变。针对可疑突变,PCR和Sanger测序对家系其他成员DNA样本进行验证。结果该家系共5代,现存家系成员38人,系谱分析符合常染色体显性遗传特征。家系共有患者17例,其临床表现为:幼时出现走路姿势异常,后出现髋关节及膝关节疼痛,X线有典型骨骺发育不良病理改变。高通量测序及数据分析后,筛选出先证者(Ⅳ-3)软骨低聚物基质蛋白(cartilage oligomeric matrix protein,COMP)基因c.1153G>A(p.Asp385Asn)错义杂合突变,该突变导致其编码蛋白的第385位天冬氨酸被天冬酰胺替代。先证者家系其他成员符合基因型与表型共分离。结论COMP基因c.1153G>A错义杂合突变是导致该MED家系患者发病的分子机制,该突变首次在大家系中被报道,进一步明确了COMP基因c.1153G>A突变的致病性,有利于家系患者的进一步的诊治,也为产前诊断提供了实验依据。     

Alkali-labile sites in sperm cells from Sus and Ovis species

Constitutive alkali-labile sites (ALSs) have been investigated using a protocol of DNA breakage detection-fluorescence in situ hybridization (DBD-FISH) in sperm cells from Sus domesticus (pig), Ovis gmelini musimon (mouflon) and Ovis aries (sheep). The results were compared with those obtained using leucocytes from the same species. Whole comparative genomic hybridization (W-CGH) showed that most of the constitutive ALSs in somatic and germ line cells in all species examined were constrained to particular repetitive satellite DNA sequences located in the pericentromeric constitutive heterochromatin of each chromosome. However, their relative abundance was different among cells of the same organism (leucocytes/sperm cells), and this trend was not maintained when the different species were compared. Thus, in mouflon, the density of ALSs in leucocytes when compared with that observed in sperm cells indicated abundance of the order of eight times less. In sheep, both leucocytes and sperm cells exhibited a large quantity of ALSs, being of the order of four times more abundant in sperm cells. In the pig genome, leucocytes showed a high abundance of ALSs (of the order of 12 times more that in sperm cells) but only involved the metacentric chromosomes of the karyotype. ALSs were not present in the acrocentric chromosomes. Contrary to mouflon and sheep, ALSs were relatively scarce in sperm cells from pig. These results suggest that ALSs are a transient structural feature in the cells of any organisms and point to a non-universal model of chromatin organization in sperm cells among mammals.     

DNA甲基化与前列腺癌

DNA甲基化是恶性肿瘤普遍存在的分子生物学改变，与肿瘤的发生和发展密切相关。前列腺癌中存在多种基因的甲基化，涉及到DNA损伤修复、激素应答、肿瘤细胞入侵／转移、细胞周期调控等通路。前列腺癌前病变如前列腺上皮内瘤也存在DNA甲基化，但程度相对较低。DNA甲基化的研究为前列腺癌的早期诊断、预后评估及激素非依赖性前列腺癌的治疗提供了新的途径。     

Sperm nuclear histone H2B： correlation with sperm DNA denaturation and DNA stainability

Aim： To examine the relationship between sperm DNA damage and sperm nuclear histone （H2B） staining. Methods： We evaluated sperm samples from 14 consecutive asthenoteratozoospermic infertile men and six consecutive fertile controls. Sperm nuclear histone （H2B） staining and sperm chromatin integrity （assessed by sperm chromatin structure assay and expressed using the percentage of （i） DNA fragmentation index [%DFI] and （ii） high DNA stainability [%HDS）]） were evaluated. Results： Histone H2B immunocytochemistry demonstrated two nuclear staining patterns： （i） focal punctate staining; and （ii） diffuse staining. Infertile men had a higher mean percentage of spermatozoa exhibiting diffuse H2B staining than did fertile men （7.7%± 4.6% vs. 1.6% ±1.2%, respectively, P 〈 0.01）. We observed significant relationships between the proportion of spermatozoa with diffuse nuclear histone staining and both sperm %DFI （r = 0.63, P 〈 0.01） and sperm %HDS （r = 0.63, P 〈 0.01）. Conclusion： The data demonstrate that infertile men have a higher proportion of spermatozoa with diffuse histone H2B than do fertile men and suggest that sperm DNA damage might, at least in part, be due to abnormally high histone H2B levels.     

纳米粒子载体携带反义转化生长因子-β1基因的局部定位转染对大鼠自体移植静脉内膜增生的影响

目的探讨纳米粒子携带反义转化生长因子(TGF-)β1局部定位转染对大鼠自体移植静脉内膜增生的影响。方法45只大鼠制成自体静脉移植模型后,分成纳米粒子DNA组、纳米粒子空载体组和生理盐水对照组,进行局部定位转染,2周后观察移植静脉内膜的变化,应用逆转录聚合酶链反应(RTPCR)、Westernblot以及免疫组织化学等方法检测反义基因对内源性TG-Fβ1表达的影响。结果基因转染后两周,内膜厚度为(20.5±4.7)μm,明显小于两个对照组(P<0.01)。RTPCR和Westernblot结果表明,基因转染组的TGF-β1mRNA和蛋白的表达明显低于空载体组及生理盐水对照组。结论纳米粒子可有效地作为基因的转移载体;纳米粒子携带反义TGF-β1可以减少内源性TGF-β1基因的表达,抑制细胞外基质(ECM)的合成,从而达到抑制内膜增生的目的。     

外周血浆K-ras基因突变的检测在胰腺癌诊断中的作用

目的 探讨检测K－ras基因突变在胰腺癌诊断中的作用。方法 收集15例胰腺癌,33例非胰腺癌病人的外周血标本,提取血浆DNA。采用限制性酶切片段长度多态性- 多聚酶链反应（RFLP－PCR）方法检测突变的K－ras基因,直接测序方法测出K－ras基因的突变类型。结果 胰腺吕病人外周血浆K-ras基因突变率为73％（11／15）,非胰腺癌组中有2例胰头肿块型慢性胰炎患者发生K-ras基因突变,其余均为阴性;血浆K－ras基因突变与肿瘤部位、大小及分期无关。结论 检测外周血浆K－ras基因突变可用于胰腺癌的辅助诊断和胰腺癌高危人群的筛选。     

TGFα在胰腺癌中的表达及其与癌细胞DNA倍型的关系

目的 研究胰腺癌中TGFα的表达及其与癌细胞DNA倍型的关系。方法 分别采用TGFα单抗免疫染色及FeuIgen染色法对67例胰腺导管腺癌和16例正常胰腺进行染色,并分析TGFα表达与癌细胞DNA倍型的关系。结果 胰腺癌组织TGFα染色阳性率为41.8%,正常胰腺组织中未见阳性染色。TGFα阳性胰腺癌癌细胞异倍体率为89.3%,明显高于阴性组。结论 TGFα表达与胰腺癌发生及癌细胞核增殖活性有关。