磷脂酰肌醇-3激酶在石英诱导的DNA双链断裂修复中的作用

目的 探讨磷脂酰肌醇-3激酶(phosphatidylinositol 3 kinase,PI3K)在石英致人胚肺成纤维细胞(HELF)DNA双链断裂修复中的作用.方法 用200μg/ml的石英刺激HELF和用显性失活突变体抑制P13K功能的HELF(DN-Δp85)不同时间.免疫印迹法检测磷酸化H2AX(γH2AX)的水平以及DNA依赖性蛋白激酶(DNA-dependent protein kinase,DNA-PK)的组成成分Ku70、Ku8和DNA-PKcs的蛋白水平,并用Image-Pro plus 6.0软件对条带光强度进行半定量分析.中性彗星试验检测DNA双链断裂损伤,用彗尾DNA百分含量值观察DNA双链断裂损伤程度变化,并计算DNA修复能力.结果 γH2AX的水平在石英刺激3 h明显增高,12 h达峰值,24 h下降.与HELF相比,石英诱导的DN-Δp85细胞γH2AX水平增高受到抑制.石英刺激HELF和DN-ΔP85细胞12 h组Ku70、Ku80和DNA-PKcs的蛋白水平(0.58±0.09、0.95±0.21、0.55±0.06,0.37±0.14、0.55±0.17、0.52±0.07)均高于相应的无石英刺激组(0.26±0.10、0.69±0.26、0.43±0.11,0.11±0.07、0.27±0.14、0.39±0.07),差异有统计学意义(P<0.05).与石英刺激HELF 12 h组比较,石英刺激DN-ΔP85 12 h组的Ku70、Ku80蛋白水平增高受到抑制,差异均有统计学意义(P<0.05).石英刺激的HELF12 h组和DN-Δp85 12h组的彗尾DNA百分含量分别为9.78±1.15和11.79±4.90,明显高于同细胞系的无石英刺激组(2.40±0.69,3.31±1.35),差异有统计学意义(P<0.05);与石英刺激HELF 12 h组相比,石英刺激HELF细胞24 h组彗尾DNA百分含量明显降低(4.19±0.47),差异有统计学意义(P<0.05).石英刺激DN-Δp85 24 h组的彗尾DNA百分含量为(7.58±4.32),明显高于无石英刺激DN-Δp85组和石英刺激HELF 24 h组,差异有统计学意义(P<0.05).HELF的DNA修复能力为75.74%,DN-Δp85的DNA修复能力为49.64%.结论 石英可诱导DNA双链断裂损伤,PI3K与DNA损伤修复有关,通过调节Ku70和Ku80的水平,可促进石英诱导的DNA双链断裂损伤的修复.     

DNA-PKcs在石英诱导的DNA双链断裂修复中的作用

目的 探讨DNA依赖性蛋白激酶催化亚单位(DNA-dependent protein kinase catalytic subunit,DNA-PKcs)在石英致人胚肺成纤维细胞(HELF)DNA双链断裂修复中的作用.方法 脂质体转染法建立DNA-PKcs的小干扰RNA(siRNA)及其阴性对照重组质粒稳定转染HELF系(简称HELF-PKcs和HELF-NC).3种方式分组及对细胞的处理:(1)25、50、100、200、300、400μg/ml浓度的石英刺激HELF 12 h;(2)200μg/ml的石英刺激HELF 0、1、2、6、12、24 h;(3)200μg/ml的石英刺激HELF-PKcs和HELF-NC 0、12、24 h.用免疫印迹法检测DNA-PKcs表达、磷酸化H2AX(H2AX)的水平.用Image-Pro plus6.0软件对条带光强度进行半定量分析;用中性彗星实验(彗尾DNA百分含量值)判断石英诱导的DNA舣链断裂损伤强度.结果 不同浓度的石英刺激HELF 12 h,γH2AX水平及彗尾DNA百分含量随着石英浓度的增加逐渐升高.200 μg/ml的石英刺激HELF6 h时,彗尾DNA百分含量[(38.7±6.9)%]与对照组相比明显增加,并且在12h达峰值,24h相对12h时点明显降低,差异均有统计学意义(P<0.05).在抑制DNA-PKcs的表达的细胞,12 h时,石英刺激的HELF-PKcs的石英诱导的γH2AX水平增加受抑制,彗尾DNA百分含量为(43.09±3.68)%,与石英刺激的HELF-NC相比,差异无统计学意义(P>0.05);24 h时,石英刺激的HELF-PKcs的石英诱导的γH2AX水平与石英刺激的HELF-NC相比无明显筹异,差异无统计学意义(P>0.05).石英刺激的HELF-PKcs的彗尾DNA百分含量(35.79±4.26)%]明显高于石英刺激的HELF-NC,差异有统计学意义(P<0.05).结论 石英可诱导DNA双链断裂损伤,DNA-PKcs是石英诱导的DNA双链断裂损伤的感受器,通过磷酸化H2AX,促进石英诱导的DNA双链断裂损伤的修复.     

名词小词典

γH2AXγH2AX是染色体组蛋白H2A家族的成员之一。在各种理化因素刺激下,细胞DNA双链发生断裂,ATM、ATR等磷脂酰肌醇3激酶使H2AX上的第139位丝氨酸发生磷酸化修饰,形成磷酸化的H2AX,即γH2AX。γH2AX募集与DNA损伤修复、细胞周期阻滞、凋亡有关的蛋白,如P53和BRCA1,形成γH2AX焦点,修复DNA损伤,引起细胞周期阻滞,诱发凋亡。因此,γH2AX可作为DNA双链断裂的早期标记物。反应时间(responsetime)是指从刺激的出现到第一个反应开始之间所经过的时间间隔,又称反应时或反应的潜伏期。反应时分为简单反应时和选择反应时两种。简…     

铅致人淋巴细胞DNA双链断裂作用的人群调查和体外试验

目的 用γH2AX识别抗体流式细胞术研究铅暴露致人体外周血淋巴细胞DNA双链断裂(DSBs)作用.方法 选取某蓄电池厂工人36人为铅接触组,其中高浓度组15人,低浓度组21人;同时选择厂外无职业性铅接触70人为对照组,取外周静脉血提取淋巴细胞,利用流式细胞术检测γH2AX,分析淋巴细胞中DNA DSBs水平;不同剂量、时间下醋酸铅染毒健康人外周血淋巴细胞,利用流式细胞术检测γH2AX,分析淋巴细胞中DNA DSBs水平.结果 高浓度铅接触组DNA损伤率和平均荧光强度分别为41.76％±28.57％、9.90±3.35,低浓度铅接触组分别为33.18％±30.64％、9.39±4.83,均高于对照组(分别为0.28％±0.28％、6.95±2.93),差异均有统计学意义(P＜0.05).体外试验结果显示,染毒1和2h时,除62.5 μmol/L外,125.0、250.0、500.0 μmol/L醋酸铅染毒组DNA损伤率与阴性对照组的差异均有统计学意义(P＜0.01).随着染毒剂量的增高,DNA损伤率呈现先增高后降低的趋势.结论 铅可致人淋巴细胞DNA DSBs,流式细胞术检测γH2AX是一种值得运用于检测大样本DNA DSBs水平的方法.     

名词小词典：γH2AX

γH2AX 是染色体组蛋白H2A家族的成员之一。在各种理化因素刺激下，细胞DNA双链发生断裂，ATM、ATR等磷脂酰肌醇3-激酶使H2AX上的第139位丝氨酸发生磷酸化修饰，形成磷酸化的H2AX，即γH2AX 。γH2AX 募集与DNA损伤修复、细胞周期阻滞、凋亡有关的蛋白，     

p53表达在石英诱导的细胞周期改变及DNA损伤修复中的作用

目的 探讨p53表达在石英致人胚肺成纤维细胞(HELF)细胞周期改变及DNA双链断裂(DNA double strand breaks,DSBs)修复中的作用.方法 3种方式分组处理细胞:(1)不同浓度(0、25、50、100、200、300、400μg/ml)的石英刺激HELF细胞12 h;(2)200 μg/ml石英刺激HELF细胞O、1、2,6、12、24 h;(3)200μg/ml石英刺激H-CMV和H-p53细胞O、12、24 h.用中性彗星试验检测石英所致的DSBs损伤强度,并计算DNA修复能力(DNA repair compentence,DRC),用免疫印迹法检测蛋白表达和磷酸化水平,用流式细胞仪检测细胞周期的变化.结果 (1)200μg/ml的石英作用HELF细胞不同时间,p53表达及p53-Ser15磷酸化水平随着作用时间的延长逐渐升高,12 h达峰值,24 h较12 h略有降低;不同浓度的石英作用HELF细胞12 h,随着石英浓度的增加,p53表达及p53-Ser15磷酸化水平逐渐增强,呈现剂量-反应关系.(2)石英作用于p53 siRNA的阴性对照细胞H-CMV,DRC为57.19%:石英作用沉默p53表达的H-p53细胞后,DSBs的修复能力增强,DRC为87.68%.(3)石英作用p53siRNA的阴性对照细胞24 h后,S期细胞比例由(24.3±3.8)%增加到(31.8±1.1)%,差异有统计学意义(P<0.05);沉默p53表达后,石英诱导的HELF细胞S期细胞比例[(41.4±0.6)%]与同期对照组[(25.4±1.9)%]相比有明显增加.差异有统计学意义(P<0.05).结论 石英可诱导p53表达及磷酸化水平的增加,p53表达上调可抑制石英所致S期细胞百分比的增加及石英所致DNA双链断裂的修复.     

DNA修复抑制状态下苯并(a)芘对人胚肺成纤维细胞的DNA损伤作用

目的研究DNA修复抑制状态下苯并(a)芘[B(a)P]对人胚肺成纤维细胞(HELF)的DNA损伤情况,探讨DNA修复机制在外源性化学致癌物诱导真核细胞DNA损伤中的作用。方法代谢活化条件下,以阿糖胞苷(ara-C,0、100μmol/L)与B(a)P(0、10、20、50μmol/L)按2×4联合染毒2h,通过彗星试验观察不同染毒条件下HELF细胞的DNA损伤情况。同时设阴性对照组(0.1%DMSO)和阳性对照组(10μmol/LK2CrO7)。结果与阴性对照组比较,B(a)P各剂量组的HELF的拖尾率、Olive尾矩随剂量增加而显著升高,差异有统计学意义(P<0.01)。100μmol/Lara-C单独染毒组与阴性对照组比较,拖尾率、Olive尾矩升高,差异有统计学意义(P<0.05,P<0.01)。B(a)P ara-C组与相应剂量的B(a)P组比较,其拖尾率、Olive尾矩升高,差异有统计学意义(P<0.01)。析因分析表明,ara-C与B(a)P诱导HELF的DNA损伤存在交互作用(F=3.219,P=0.024)。ara-C可增强B(a)P对HELF的DNA损伤作用。结论DNA修复抑制在外源性化学致癌物诱导真核细胞DNA损伤中存在重要作用。     

1800 MHz射频电磁场对中国仓鼠肺成纤维细胞DNA损伤的影响

目的研究全球移动通讯系统(GSM)1800 MHz射频电磁场对中国仓鼠肺成纤维细胞(CHL)DNA损伤的影响.方法采用DNA双链断裂的早期标志性事件H2AX的磷酸化作为检测指标,将细胞间断(5 min开,10 min关)暴露于比吸收率为0或3.0 W/kg的1800 MHz射频电磁场1或24 h后,用4%多聚甲醛固定后进行γH2AX免疫荧光分析,即用鼠源抗γH2AX单克隆抗体为一抗和异硫酸酯荧光素标记的山羊来源抗鼠抗体为二抗,显示细胞核内γH2AX的形成情况.以4,6-二咪基-4-联苯基吲哚标记细胞核,采用Image Pro-Plus图像软件分析数据,以20 mg/L的DNA损伤剂二乙酰氨基芴(AAF)作用2 h作为阳性对照.各处理条件至少检测50个细胞的γH2AX 焦点数,焦点超过5个的细胞定义为γH2AX焦点阳性细胞,将该焦点阳性细胞率作为评价细胞DNA损伤程度的指标.结果γH2AX焦点阳性细胞率在1800 MHz射频暴露24 h后为(37.9±8.6)%,在阳性对照组为(50.9±9.4)%,与假辐照组的(28.0±8.4)%比较,明显增高;而射频暴露1 h后的γH2AX焦点阳性细胞率为(31.8±8.7)%,增加不明显.结论 1800 MHz 比吸收率为3.0 W/kg的射频电磁场辐照24 h对CHL细胞DNA有损伤作用.     

二甲基甲酰胺致人肝细胞DNA的损伤效应

目的 探讨不同染毒剂量二甲基甲酰胺(DMF)在不同染毒时间下对人正常离体肝细胞DNA损伤的影响.方法 肝细胞染毒分别以PBS阴性对照组、1.56、6.25、25.00、100.00 mmol/L的DMF和100μmol/L的H2O2,染毒时间为0.5、3.0、6.0、12.0和24.0 h,用γH2AX免疫荧光法观察肝细胞DNA的损伤情况.结果 不同剂量的DMF和H2O2诱导形成γH2AX焦点的细胞数量多于PBS阴性组,差异有统计学意义(P＜0.01),并且随着DMF染毒剂量的增加,形成γH2AX焦点细胞数有增多趋势;在不同染毒时间处理后,γH2AX焦点细胞数的形成除3.0 h组和0.5 h组比较,差异无统计学意义(P＞0.05),余处理时间组与0.5 h比较,差异均有统计学意义(P＜0.01),并且形成γH2AX焦点细胞数有减少趋势.结论 不同剂量DMF可引起DNA的损伤,并且损伤程度与剂量呈正相关,与染毒时间呈负相关,γH2AX可作为检测细胞DNA损伤程度的良好指标.     

甲醛暴露工人外周血淋巴细胞遗传物质损伤水平的研究

目的评价甲醛暴露工人外周血淋巴细胞DNA和染色体损伤水平。方法选择2家密度板厂151名甲醛暴露工人(暴露组)和某推土机厂112名非甲醛暴露工人(对照组)为研究对象,用气相色谱法检测作业环境的甲醛浓度,采用彗星试验和胞质分裂阻滞微核试验,评价其外周血淋巴细胞DNA和染色体损伤水平。结果暴露组工人作业环境中的甲醛8h时间加权平均浓度(0.10～7.88mg/m3)高于对照组(<0.01mg/m3)。高暴露组Olive尾矩为3.95,95%CI为3.53～4.43;低暴露组Olive尾矩为3.03,95%CI为2.49～3.67;对照组Olive尾矩为0.93,95%CI为0.78～1.10,各组间差异均有统计学意义。暴露组彗星尾长为6.78,95%CI为6.05～7.60,均高于对照组,差异有统计学意义。高暴露组彗星尾长为12.59,95%CI为11.80～13.43;低暴露组彗星尾长为11.25,95%CI为10.12～12.50,差异无统计学意义。高暴露组的微核率为(0.65±0.36)%,低暴露组的微核率为(0.41±0.25)%,对照组的微核率为(0.27±0.13)%,各组间的差异均有统计学意义。各指标均有随暴露水平增加而增高的趋势,按吸烟情况分层后仍有增高的趋势。结论甲醛暴露可导致工人外周血淋巴细胞的DNA和染色体损伤水平增高,并且有随暴露水平增加而增高的趋势。     

Calcium intake in the elderly: validation of a dietary questionnaire

Epidemiological studies of calcium and osteoporosis have been hampered by the lack of a suitable tool for assessing calcium intake. This report describes a new frequency and amount questionnaire for measuring present and past calcium intake in the elderly. The validity of the questionnaire was tested against two commonly used standards of dietary assessment, five-day duplicate diets and seven-day weighed dietary inventories. The resulting correlation coefficients were, respectively, r = 0.76 and r = 0.69, while that for repeatability was r = 0.84. Furthermore, the questionnaire categorized subjects into thirds of the distribution of intake with almost no gross misclassification. It is suggested that the present findings may be extended to the majority of normal, healthy elderly subjects, implying wide application for the questionnaire in the assessment of calcium intake in the elderly.     

The occurrence of antimicrobial substances in toilet,sink and shower drainpipes of clinical units: A neglected source of antibiotic residues

Antibiotics represent one of the most important drug groups used in the management of bacterial infections in humans and animals. Due to the increasing problem of antibiotic resistance, assurance of the antibacterial effectiveness of these substances has moved into the focus of public health. The reduction in antibiotic residues in wastewater and the environment may play a decisive role in the development of increasing rates of antibiotic resistance. The present study examines the wastewater of 31 patient rooms of various German clinics for possible residues of antibiotics, as well as the wastewater of five private households as a reference.To the best of our knowledge, this study shows for the first time that in hospitals with high antibiotic consumption rates, residues of these drugs can be regularly detected in toilets, sink siphons and shower drains at concentrations ranging from 0.02?μg·L^?1 to a maximum of 79?mg·L^?1. After complete flushing of the wastewater siphons, antibiotics are no longer detectable, but after temporal stagnation, the concentration of the active substances in the water phases of respective siphons increases again, suggesting that antibiotics persist through the washing process in biofilms. This study demonstrates that clinical wastewater systems offer further possibilities for the optimization of antibiotic resistance surveillance.     

Self-reported health problems among Swedish miners one year after unemployment

Unemployment is considered to be a public health concern sincedeterioration in the health of the unemployed is often anticipated.However, for some groups, such as miners, unemployment mightimprove health due to a cessation of potentially harmful occupationalexposures. This study evaluates the health of 79 miners in oneSwedish iron-ore mine, and 226 age-matched controls from thegeneral population, during one year after the closure of themine. The participants received a questionnaire regarding medicalhistory and subjective symptoms at the beginning of the studyperiod, and after one year. Statistically significant negativeeffects on self-reported health attributable to unemploymentwere not found, although neuropsychiatric symptoms were morecommon among the unemployed miners. The miners reported a statisticallysignificant improvement in grip force (p=0.031). They had asignificantly higher prevalence of symptoms associated withmining related exposures when compared with the population controls;pain in the upper extremities [relative risk (RR)=2.27, 95%confidence interval (Cl)=1.44–3.59), back pain (RR=1.84;Cl=1.237–2.75), vasospastic disease of the fingers (RR=2.05;Cl=1.18–3.57) and obstructive respiratory symptoms (attacksof dyspnea and wheezing: RR=3.67; Cl=1.167–11.6).     

Excès de cas humains et animaux de tularémie en France au cours de l’hiver 2007–2008 : émergence ou phénomène isolé ?

Context

Tularemia is a zoonosis affecting humans and hares in France. We describe the results of surveillance in both species, in 2007 and 2008.

Methods

Human tularemia cases are mandatorily notifiable in France since 2003. In hares, surveillance relies on volunteer hunter associations in all districts of the country. Data from mandatory reports and volunteer surveillance in 2007/2008 were analyzed and compared with previous results.

Results

In 2007/2008, 144 cases were reported in humans and 117 cases in hares. This was a 100% increase compared to previous years. Human cases differed from those of previous years only by the frequency of contact with breeding animals. Human cases without any documented risk exposure were also more frequent.

Conclusion

An increase of tularemia cases occurred in 2007/2008 in both species. Complementary studies are needed to identify the species reservoir in France to understand the causes of this peak of cases.     

Occupational health hazards in mining: an overview

This review article outlines the physical, chemical, biological, ergonomic and psychosocial occupational health hazards of mining and associated metallurgical processes. Mining remains an important industrial sector in many parts of the world and although substantial progress has been made in the control of occupational health hazards, there remains room for further risk reduction. This applies particularly to traumatic injury hazards, ergonomic hazards and noise. Vigilance is also required to ensure exposures to coal dust and crystalline silica remain effectively controlled.     

Manufacture and use of nanomaterials: current status in the UK and global trends

This paper provides an overview of the production and use of nanomaterials (NMs), particularly in the UK. Currently, relatively few companies in the UK are identifiable as NM manufacturers, the main emphasis being the bulk markets in metals and metal oxides, and some niche markets such as carbon nanotubes and quantum dots. NM manufacturing in the UK does not reflect the global emphasis on fullerenes, nanotubes and fibres. Some assumptions have been made about the types of NM that are likely to be imported into the UK, which currently include fullerenes, modified fullerenes and other carbon-based NMs including nanotubes. Many university departments, spin-offs and private companies have developed processes for the manufacture of NMs but may only be producing small quantities for research and development (R&D) purposes. However, some have the potential to scale up to produce large quantities. The nanotechnology industry in the UK has strong R&D backup from universities and related institutions. This review has covered R&D trends at such institutions, and appropriate information has been added to a searchable database. While several companies are including NMs in their products, only a few (e.g. manufacturers of paints, coatings, cosmetics, catalysts, polymer composites) are using nanoparticles (NPs) in any significant quantities. However, this situation is likely to change rapidly. There is a need to collect more information about exposure to NPs in both manufacturing and user scenarios. As the market grows, and as manufacturers switch from the micro- to the nanoscale, the potential for exposure will increase. More research is required to quantify any risks to workers and consumers.     

深圳公立医院管理体制改革制度设计

深圳公立医院管理体制改革实行政事分开、管办分开,在理事会架构下按法定机构模式组建市医管中心,落实公立医院运营管理自主权.作者从当前公立医院管理体制的弊端入手,介绍了深圳市进行公立医院管理体制改革的基本思路及改革方案设计的主要举措,深入剖析了的改革方案的特点,并对改革效果进行了预测.     

Effects of nitrogen dioxide on red blood cells of rats: Changes in components of red cell membranes during in vivo exposure to NO2

Red cell membranes, prepared from red blood cells of rats exposed to 4, 10, or 20 ppm nitrogen dioxide (NO₂) for 1 to 10 days, were examined for evidence of changes in membrane components. Appreciable changes were not found in contents of phospholipid and cholesterol during exposure to 10 ppm NO₂. By contrast, protein content altered with the time of exposure. Moreover, changes in protein composition were observed by employing sodium dodecyl sulfate — polyacrylamide gel electrophoresis. Twenty-four-hour exposure to NO₂ at the concentration above 10 ppm resulted in a marked increase in the percentage of lysophosphatidylethanolamine (LysoPE) to the total phospholipids. The prolonged exposure to 10 ppm NO₂ gave rise to a further increase in LysoPE, whereas the percentage of phosphatidylethanolamine (PE) showed a gradual decrease. A 1-day exposure to 4.0 ppm NO₂ also caused an increase in sialic acid content and decreases in those of PE and hexose. In addition to contents of these components the percentage of LysoPE increased 5 days after exposure and the elevated values were maintained up to the end of exposure period. These results demonstrate that red blood cells in circulation exhibit different membrane properties in terms of lipid and carbohydrate composition during 10 days of exposure to 4.0 ppm NO₂.     

Investigating clusters in the workplace and beyond

Clusters of disease are common and occur in the workplace and in the general community. They often arouse considerable concern among the population. Investigations have sometimes lead to exciting new knowledge, but in general the investigation of clusters is difficult and often unrewarding, especially for community clusters. In the workplace, investigations are more likely to find associations and even new causes, but still many clusters remain enigmatic. Despite this, there are many reasons for investigating clusters, including allaying community concern and identifying uncontrolled exposures. A structure for investigating clusters in the workplace is suggested.