MicroRNA-27a通过靶向HBx mRNA抑制HBV复制

目的　探究微小RNA-27a（microRNA-27a, miR-27a）在HBV感染中的作用及分子机制。方法　实时定量PCR（quantitative real-time PCR, qRT-PCR）方法检测miR-27a对HBV DNA和HBV RNA的影响。ELISA法检测miR-27a对HBeAg和HBsAg的影响。生物信息学预测miR-27a的靶基因,然后用双荧光素酶报告基因检测、qRT-PCR和western blot检测miR-27a是否能够直接靶向靶基因以及对靶基因mRNA和蛋白表达的影响。最后用qRT-PCR检测HBV蛋白表达对miR-27a的影响。结果　MiR-27a能抑制HBV DNA、HBV RNA、HBeAg和HBsAg等HBV复制和表达的指标。生物信息学预测和双荧光素酶报告基因检测表明miR-27a能够直接靶向HBx,抑制HBx mRNA和蛋白表达。反之,HBx蛋白表达能抑制细胞内miR-27a的表达。结论　MiR-27a通过直接靶向HBx进而抑制HBV的复制和表达,可以作为一种新的治疗HBV感染的潜在靶点。     

miR-15-5p靶向调控SMAD5表达抑制子宫内膜腺癌细胞上皮间质转化的研究

目的探讨miR-15-5p抑制人子宫内膜腺癌细胞(HEC-1-A)上皮间质转化(EMT)的机制。方法构建SMAD53'-UTR-荧光素酶报告载体,通过双荧光素酶报告基因检测,观察miR-15-5p对SMAD53'-UTR荧光素酶活性的影响,鉴定SMAD5是否为miR-15-5p的靶基因。将miR-15-5p模拟物(agomir)与miR-15-5p抑制物(antagomir)及其对应的对照组分别转染至HEC-1-A细胞中,并进行EMT诱导培养,分别采用实时荧光定量PCR(qRT-PCR)及Western Blot检测各组细胞SMAD5在mRNA及蛋白水平的相对表达量;了解miR-15-5p对EMT标志物的表达影响。结果双荧光素酶报告基因实验结果表明,miR-15-5p可以结合在SMAD5的3'-UTR区域,而不能结合在SMAD3的3'-UTR区域,表明miR-15-5p的靶基因应为SMAD5。正常HEC-1-A细胞转染miR-15-5p的模拟物或抑制物后,SMAD5表达显著下调或上调,也表明miR-15-5p的靶基因是SMAD5。结论SMAD5是miR-15-5p的靶基因,miR-15-5p可通过靶向调控SMAD5的表达抑制人子宫内膜腺癌HEC-1-A细胞的EMT。     

沉默lncRNA MAFG-AS1对人卵巢癌细胞增殖和凋亡的影响及分子机制

目的探讨沉默lncRNA MAFG-AS1对人卵巢癌细胞增殖和凋亡的影响及分子机制。方法 qRT-PCR检测正常卵巢上皮细胞HOSE和3种卵巢癌细胞(SKOV3、HO8910、OVCAR3)中MAFG-AS1和miR-143-3p的表达。荧光素酶报告基因检测和qRT-PCR验证MAFG-AS1与miR-143-3p的靶向调控关系。以SKOV3细胞为研究对象,分别构建沉默MAFG-AS1或过表达miR-143-3p的卵巢癌细胞株。应用MTT法检测细胞活力,应用流式细胞术检测细胞凋亡情况,应用Western Blot检测增殖相关蛋白Cyclin D1和p21及凋亡相关蛋白Bcl-2及Bax的表达。结果与HOSE组比较,3种卵巢癌细胞中MAFG-AS1的表达显著上调,miR-143-3p的表达显著下调。pc DNA组、pc DNA-MAFG-AS1组、si-NC组、si-MAFG-AS1组miR-143-3p表达水平比较差异有统计学意义(P0.05)。miR-143-3p是MAFG-AS1的靶基因,MAFG-AS1可负性调控miR-143-3p的表达。沉默MAFG-AS1或过表达miR-143-3p均可抑制卵巢癌细胞的增殖,促进细胞凋亡,抑制Cyclin D1和Bcl-2蛋白表达,促进p21和Bax蛋白表达。抑制miR-143-3p表达可逆转沉默MAFG-AS1对卵巢癌细胞的增殖抑制和凋亡促进作用。结论沉默MAFGAS1通过上调miR-143-3p表达可抑制卵巢癌细胞的增殖,促进细胞凋亡。     

异丙酚调控miR-212-5p/TRPV6轴对卵巢癌细胞增殖 迁移及侵袭影响的机制研究

目的探讨异丙酚通过调控miR-212-5p/TRPV6轴对卵巢癌细胞增殖、迁移和侵袭影响和机制,为临床诊治卵巢癌奠定理论基础。方法不同浓度的异丙酚[(0μM (μmol/L)、0.125μM、0.25μM、0.5μM和1.0μM]作用SKOV3细胞48 h后,MTT实验检测细胞活力,Transwell实验检测细胞的迁移和侵袭能力,qRT-PCR和Western blot检测miR-212-5p、TRPV6、Cyclin D1、MMP2和MMP9蛋白的表达变化。同时,构建抑制miR-212-5p表达的SKOV3细胞株,进行上述实验检测相应指标。双荧光素酶实验和Western blot验证miR-212-5p和TRPV6的靶向关系。结果异丙酚呈浓度依赖性地抑制SKOV3细胞的增殖、迁移和侵袭,抑制TRPV6、Cyclin D1、MMP2和MMP9蛋白的表达,促进miR-212-5p的表达。抑制miR-212-5p表达可减轻异丙酚处理对SKOV3细胞的增殖、迁移和侵袭的抑制作用。TRPV6是miR-212-5p的靶基因,miR-212-5p可负性调控TRPV6的表达。沉默TRPV6可部分逆转抑制miR-212-5p表达对异丙酚处理的卵巢癌细胞SKOV3增殖、迁移和侵袭的影响。结论异丙酚通过上调miR-212-5p表达,抑制TRPV6表达,从而抑制卵巢癌细胞增殖、迁移和侵袭。     

乙型肝炎病毒RNA在慢性乙型病毒性肝炎不同HBeAg状态下的临床研究

目的 通过观察HBeAg不同状态下慢性乙型肝炎(chronic hepatitis B,CHB)的HBV RNA表达水平、检出率分析以及与HBV DNA、HBsAg的相关性，探索血清HBV RNA在CHB管理中的价值。方法 本研究共纳入2021年1月—12月在杭州市西溪医院门诊及住院部就诊的CHB患者253例，包括HBeAg阳性的患者103例和HBeAg的阴性患者150例，检测血清HBV RNA定量，并分析其与HBsAg、HBV DNA等的相关性。结果 在HBeAg阳性组与HBeAg阴性组之间，HBV RNA及HBV DNA的表达水平，差异均有统计学意义(P<0.001)。HBeAg阳性患者的HBV RNA、HBV DNA、HBsAg、ALT、AST定量均高于HBeAg阴性患者。HBeAg阳性组中，血清HBV RNA与HBV DNA、HBsAg成正相关(r值分别为0.855、0.669,P<0.001);HBeAg阴性组中，血清HBV RNA与HBV DNA、HBsAg成正相关(r值分别为0.476、0.375,P<0.001)。在CHB未治患者中HBV RNA与HB...     

miR-424-5p靶向KIF23调控细胞周期并抑制肝癌细胞的增殖和迁移能力

目的 探究miR-424-5p、KIF23对肝癌细胞的作用机制,为探索新的肝癌靶向治疗途径提供思路。方法 实时荧光定量聚合酶链式反应(qRT-PCR)检测miR-424-5p的表达水平,生物信息学技术和萤光素酶报告基因验证KIF23与miR-424-5p在肝癌中的结合关系。qRT-PCR和蛋白免疫印迹(western blot, WB)分别检测KIF23 mRNA和蛋白表达水平。CCK8实验、克隆形成实验、迁移实验和流式细胞术评估miR-424-5p和KIF23对肿瘤细胞的增殖、迁移能力,以及对细胞周期进展的影响。结果 与正常肝组织比较,miR-424-5p在肝癌组织和细胞中显著低表达(P<0.05);KIF23是miR-424-5p的直接下游靶点,在肝癌组织和细胞中的表达显著上调(P<0.05)。过表达KIF23会促进肝癌细胞增殖和迁移,并将细胞周期阻滞于G2/M期。过表达miR-424-5p能显著减弱过表达KIF23对细胞增殖、迁移和细胞周期分布的影响(P<0.05)。结论 miR-424-5p可以通过靶向下调KIF23,从而抑制肝癌细胞增殖和迁移能力,影响细胞周...     

乙型肝炎病毒不同基因区RNA干扰抑制乙型肝炎病毒复制和表达的实验研究

目的针对乙型肝炎病毒(HBV)不同基因区设计siRNA,观察其对HBV表面抗原、e抗原分泌和HBVDNA复制的影响。方法靶向HBV基因区S、C、X的siRNA分别转染可稳定分泌HBV颗粒的HepG2.2.15细胞,转染后72h,采用ELISA方法检测上清液中HBsAg、HBeAg的含量,Real-timePCR定量检测HBVDNA拷贝数。结果靶向不同基因区的siRNA均能不同程度地抑制HepG2.2.15细胞HBsAg、HBeAg的分泌和HBVDNA的复制,其中S区siRNA对HBsAg呈现非常显著的抑制作用,抑制率为91.88%(P﹤0.01),对HBeAg表达的抑制作用较弱,抑制率为24.85%(P﹤0.05),对HBVDNA的抑制率为63.07%(P﹤0.01);C区siRNA对HBeAg和HBVDNA抑制作用较强,分别为54.77%(P﹤0.01)和76.97%(P﹤0.01),但对HBsAg的表达无明显影响(P﹥0.05);X区siRNA对HBsAg和HBeAg、HBVDNA均有抑制作用,抑制率分别为90.83%(P﹤0.01)、34.85%(P﹤0.01)和70.31%(P﹤0.01)。靶向不同基因区的siRNA对HBsAg、HBeAg、HBVDNA的抑制作用在一定的程度上存在剂量效应,而无关序列对HBsAg、HBeAg和HBVDNA的复制和表达几乎无干扰作用(P﹥0.05)。结论靶向HBVC、S、X基因区的siRNA可特异、高效、稳定地抑制HBV的复制和表达,为应用RNA干扰治疗乙型肝炎病毒奠定了一定基础。     

RIF患者血浆外泌体源miRNA-183-5p通过靶向FOXO1影响蜕膜化

目的 探讨反复种植失败(RIF)患者血浆外泌体源性miR-183-5p通过靶向FOXO1影响子宫内膜基质细胞蜕膜化。方法 选取2021年8月1日至2021年12月31日在空军军医大学唐都医院生殖中心就诊的不孕患者,其中接受IVF治疗的20名RIF患者作为RIF组,20名同期无特殊病史的不孕患者作为对照组。采用实时荧光定量聚合酶链反应(qRT-PCR)检测两组不孕患者的血浆外泌体miR-183-5p的表达。从血浆中提取外泌体并与人子宫内膜间质细胞系(HESC)共培养48h后进行蜕膜化诱导,检测人胰岛素样生长因子结合蛋白-1(IGFBP-1)和催乳素(PRL)的表达。将miR-183-5p的agomir和antagomir转染HESC细胞并诱导蜕膜化后检测蜕膜化水平。通过qRT-PCR和免疫蛋白印迹(WB)实验检测miR-183-5p对于FOXO1 mRNA和蛋白表达水平的影响。结果 与对照组比较,RIF患者血浆外泌体能够抑制蜕膜化的子宫内膜基质细胞IGFBP-1和PRL的表达(t值分别为5.324、9.056,P<0.05)。与对照组比较,RIF患者血浆外泌体和子宫内膜组织高表达miR-183-5p((t值为-2.486、-4.821,P<0.05)。miR-183-5p能够抑制蜕膜化HESC细胞FOXO1的mRNA(agomir-NC vs agomir,t=3.120,P<0.05)和蛋白质的表达(对照组vs agomir,t=2.793,P<0.05)。结论 RIF患者血浆外泌体源性miR-183-5p能够抑制FOXO1表达,从而影响子宫内膜基质细胞蜕膜化。     

HepG2-hNTCP细胞模型的构建及在miRNA-548ah调控乙型肝炎病毒研究中的应用

目的构建并鉴定表达人钠离子-牛磺胆酸共转运蛋白(hNTCP)的HepG2-hNTCP细胞模型,探讨微小RNA-548ah (miR-548ah)对乙型肝炎病毒复制和表达的影响。方法扩增重组慢病毒pWPI-hNTCP-Puro质粒,pWPI-hNTCP-Puro慢病毒感染HepG2细胞,聚合酶链反应(PCR)检测hNTCPmRNA的表达水平,共聚焦显微镜检测hNTCP蛋白定位。Lipofectamine转染miR-548ah激动剂、miR-548ah抑制剂及阴性对照至细胞系。酶联免疫吸附测定检测HBsAg和HBeAg的表达水平,荧光定量PCR检测HBVDNA的表达,应用SPSS 17.0对数据进行分析。结果 hNTCP mRNA在HepG2hNTCP呈高水平表达。共聚焦荧光显微镜观察HepG2hNTCP中的hNTCP蛋白正常表达且主要定位在细胞膜上。与HepG2细胞比较,HBsAg和HBeAg在感染后HepG2-hNTCP细胞5天和7天后的表达水平存在差异,随培养天数呈上升趋势,具有统计学意义(P0.01)。与转染阴性对照比较,HBsAg在转染miR-548ah激动剂组的表达水平升高,差异具有统计学意义(P0.05);而HBeAg和HBVDNA的表达水平无明显变化,差异均无统计学意义。结论成功构建了表达hNTCP的HepG2-hNTCP细胞模型,对乙型肝炎病毒感染具有良好的易感性;miR-548ah可促进HBV感染HepG2-hNTCP细胞模型中HBsAg的表达。     

miR-485-5p靶向Toll样受体4调控巨噬细胞的炎症反应

目的 探究miR-485-5p对Toll样受体4(TLR-4)的靶向作用，研究其在巨噬细胞炎症反应中的调控作用。方法 合成miR-485-5p模拟物(mimics)及其抑制剂(inhibitors),分别转染HEK-293T细胞，RT-PCR检测miR-485-5p的表达水平。根据miRNA靶向作用预测数据分析选定TLR-4作为研究的靶基因，构建TLR-43’UTR及其突变体萤火虫-海肾双荧光素酶报告基因系统，通过检测在细胞模型中过表达/抑制表达miR-485-5p后对荧光素酶活力的影响，验证miR-485-5p与TLR-4之间的靶向关系，Western blot检测TLR-4蛋白质表达水平。构建巨噬细胞模型，将miR-485-5p mimics及inhibitors分别转染巨噬细胞，RT-PCR检测靶基因TLR-4 mRNA的表达水平，ELISA检测上清中下游致病因子IL-6、 IL-17、IL-18的表达。结果 与对照组比较，转染miR-485-5p mimics后miR-485-5p表达水平升高，转染miR-485-5p inhibitors后miR-485-5p表达水平降低(P...     

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.     

The human drama of genetics: 'hard' and 'soft' media representations of inherited breast cancer

This paper examines media coverage of 'breast cancer genetics', and explores its implications for public understanding. We present a content analysis of coverage in British newspapers and look at a variety of popular forms, including women's magazines, television soap opera and radio drama. Genetic/inherited risk receives a great deal of coverage across a wide range of media formats and outlets. Much of this attention has focused on individuals from 'high risk families' and dilemmas around prophylactic mastectomies. Through examining media coverage, combined with interviews with media personnel and their sources, we show why this story proved so attractive to the media and highlight the different production values which influence coverage. Finally, we introduce preliminary findings from focus group discussions to demonstrate how such 'human interest' framing has engaged audience attention and influenced public understandings. The paper concludes by highlighting the implications for analysing, predicting, and engaging with, media representations of science.     

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.     

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.     

Fatigue and health in a seafaring population

Background Occupational fatigue is relatively common withinthe general population and has been linked to reduced performance,injury and longer term ill-health. Despite growing acknowledgementof this problem in the maritime sector, little research hasbeen conducted into the risk factors, prevalence and consequencesof seafarers' fatigue. Aims To examine the prevalence of fatigue among seafarers, identifypotential risk factors and assess possible links with poor performanceand ill-health. Methods Cross-sectional questionnaire survey of seafarers workingin the offshore oil support, short-sea and deep-sea shippingindustries. A number of tools were used including the fatiguesubscale of the profile of fatigue-related symptoms, the CognitiveFailures Questionnaire, the General Health Questionnaire andthe SF36 General Health scale. Results In all, 1855 questionnaires were completed giving anoverall response rate of 20%. Fatigue symptoms were associatedwith a range of occupational and environmental factors, manyunique to seafaring. Reporting a greater number of risk factorswas associated with greater fatigue [e.g. OR = 2.53 (1.90–3.35)for those with three or four risk factors and OR = 9.54 (6.95–13.09)for those with five or more risk factors]. There was also astrong link between fatigue and poorer cognitive and healthoutcomes, with fatigue the most important of a number of riskfactors, accounting for 10–14% of the variance. Conclusions Seafarers' fatigue could impact on safety withinthe industry and may be linked to longer term individual ill-health.It can only be addressed by considering how multiple factorscombine to contribute to fatigue.     

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.