碘缺乏和甲状腺功能减退对大鼠仔鼠海马ERK1/2表达的影响

目的观察碘缺乏和甲状腺功能减退对大鼠仔鼠海马细胞外信号调节蛋白激酶1及2(ERK1/2)表达的影响。方法健康2月龄孕Wistar大鼠28只,按体重随机分成对照组、甲状腺功能减退组[按饮水中含丙基硫尿嘧啶(PTU)剂量分为5mg/L组和15mg/L组]和碘缺乏组,每组7只。分别于出生后第7、14、21、28和42天每组随机取5只仔鼠,灌流固定大脑,用组织病理切片和免疫组化染色观察分析海马的ERK1/2表达。结果在出生后14、21、28和42天时,海马CA1和CA3区的ERK1/2表达在PTU5mg/L组、PTU15mg/L组和碘缺乏组显著低于对照组(P0.05)。DG区的ERK1/2表达与对照组相比差异无显著性。出生后7天时,各组间ERK1/2表达差异无显著性。结论碘缺乏和甲状腺功能减退可降低海马CA1和CA3区的ERK1/2表达。     

上海地区224例早期新型甲型H1N1型流感病例临床特征、治疗及预后回顾性分析

目的 回顾性分析上海地区早期新型甲型H1N1型流感病例的临床特点、治疗及预后.方法 以上海市公共卫生临床中心收治的224例新型A/H1N1型流感患者为研究对象,分析患者基本信息、临床症状与体征、实验室及影像学检查、病程、抗病毒治疗后新型A/H1N1病毒核酸转阴时间及住院时间等.结果 （1）男女性别比为1.1∶1,63.8%的患者年龄分布在18～40岁之间;（2）临床表现以发热、咳嗽、咽部充血,中性粒细胞和C反应蛋白（CRP）升高为特征,且男性白细胞和CRP水平显著高于女性[分别为（6.71±2.45）× 10^9/L比（5.74±2.10）× 10^9/L,F=10.063,P=0.002;（16.32±18.27）mg/L比（12.05±11.44）mg/L,F=4.026,P=0.046];（3）女性血清前白蛋白（PA）水平显著低于男性[（244.80±76.05）mg/L比（272.06±93.51）mg/L,F=5.800,P=0.017];（4）经CT确诊为肺炎者占21.9%;（5）治疗后血清CRP水平显著降低[（3.81±4.93）mg/L比（10.80±12.92）mg/L,F=6.709,P=0.013];CD3、CD4及CD8水平显著升高[分别为（1449.46±446.71）个/μl比（700.50±307.46）个/μl,F=57.940,P=0.000;（762.69±255.66）个/μl比（370.08±157.62）个/μl,F=47.587,P=0.000;（592.38±238.24）个/μl比（288.88±164.98）个/μl,F=34.409,P=0.000];（6）新型A/H1N1型流感病程（3.9±1.4）d,治疗后病毒mRNA转阴时间为（3.8±1.3）d,住院时间（5.0±1.7）d.结论 （1）新型A/H1N1型流感临床以发热、咳嗽、咽部充血、CRP和中性粒细胞升高为特征;（2）新型A/H1N1型流感病毒具有多器官分布特性,可影响肝脏合成PA及机体的免疫功能;（3）早期新型A/H1N1型流感临床症状较轻、病程短,奥司他韦疗效肯定,预后良好.     

Apoptosis and pathogenesis of avian influenza A (H5N1) virus in humans

The pathogenesis of avian influenza A (H5N1) virus in humans has not been clearly elucidated. Apoptosis may also play an important role. We studied autopsy specimens from 2 patients who died of infection with this virus. Apoptosis was observed in alveolar epithelial cells, which is the major target cell type for the viral replication. Numerous apoptotic leukocytes were observed in the lung of a patient who died on day 6 of illness. Our data suggest that apoptosis may play a major role in the pathogenesis of influenza (H5N1) virus in humans by destroying alveolar epithelial cells. This pathogenesis causes pneumonia and destroys leukocytes, leading to leukopenia, which is a prominent clinical feature of influenza (H5N1) virus in humans. Whether observed apoptotic cells were a direct result of the viral replication or a consequence of an overactivation of the immune system requires further studies.     

p-ERK1/2在急性髓系白血病的表达及意义

目的检测磷酸化的细胞外信号调节激酶(p-ERK1/2)在急性髓系白血病(AML)中的表达,分析其与外周血白细胞数的相关性,探讨p-ERK1/2在AML发生、发展和治疗中的意义。方法选取同期收治的AML初治组患者26例(其中治疗后完全缓解者20例,未缓解者6例)、复发组患者20例、正常对照组20例骨髓细胞为研究对象,应用流式细胞仪检测p-ERK1/2的表达。结果 AML初治组及复发组p-ERK1/2的表达水平均显著高于对照组及完全缓解组(P<0.01),而完全缓解组与对照组p-ERK1/2的表达水平无显著差异(P>0.05),相关性分析表明白细胞数与p-ERK1/2的表达密切相关(P<0.01)。结论 p-ERK1/2的表达在AML发生、发展中有重要意义。     

The novel adjuvant CoVaccineHT™ increases the immunogenicity of cell-culture derived influenza A/H5N1 vaccine and induces the maturation of murine and human dendritic cells in vitro

A candidate influenza H5N1 vaccine based on cell-culture-derived whole inactivated virus and the novel adjuvant CoVaccineHT™ was evaluated in vitro and in vivo. To this end, mice were vaccinated with the whole inactivated influenza A/H5N1 virus vaccine with and without CoVaccineHT™ and virus-specific antibody and cellular immune responses were assessed. The addition of CoVaccineHT™ increased virus specific primary and secondary antibody responses against the homologous and an antigenically distinct heterologous influenza A/H5N1 strain. The superior antibody responses induced with the CoVaccineHT™-adjuvanted vaccine correlated with the magnitude of the virus-specific CD4+ T helper cell responses. CoVaccineHT™ did not have an effect on the magnitude of the CD8+ T cell response. In vitro, CoVaccineHT™ upregulated the expression of co-stimulatory molecules both on mouse and human dendritic cells and induced the secretion of pro-inflammatory cytokines TNF-α, IL-6, IL-1β and IL-12p70 in mouse- and IL-6 in human dendritic cells. Inhibition experiments indicated that the effect of CoVaccineHT™ is mediated through TLR4 signaling. These data suggest that CoVaccineHT™ also will increase the immunogenicity of an influenza A/H5N1 vaccine in humans.     

In vitro antiviral activity of hypothiocyanite against A/H1N1/2009 pandemic influenza virus

Influenza virus spreads via small particle aerosols, droplets and fomites, and since it can survive for a short time on surfaces, can be introduced into the nasal mucosa before it loses infectivity. The hypothiocyanite ion (OSCN⁻), product of the lactoperoxidase/H₂O₂/SCN⁻ system of central airways, is emerging as an important molecule for innate defense mechanism against bacteria, fungi and viruses. Here we demonstrated that OSCN⁻ displays virucidal activity in vitro against the A/H1N1 2009 pandemic influenza virus. The concentration required to inhibit viral replication by 50% was 2 μM when virus were challenged directly with OSCN⁻ before cell inoculation. These values were even lower when inoculated cells were maintained in contact with enzyme free-OSCN⁻ in the culture medium. The last experimental conditions better reflect those of tracheobronchial mucosa, where HOSCN/OSCN⁻ is retained in the air–liquid interface and inactivates both the viruses approaching the epithelium from outside and those released from the inoculated cells after the replication cycle. Importantly no OSCN⁻ cytotoxicity was observed in the cellular system employed. The lack of toxicity in humans and the absence of damage on surfaces of fomites suggest a potential use of OSCN⁻ to avoid mucosal and environmental transmission of influenza virus. Since hypothiocyanite is normally present in human airways a low risk of viral resistance is envisaged. In vivo confirmatory studies are needed to evaluate the appropriate dose, regimen and formulation.     

Chimpanzee adenovirus vector-based avian influenza vaccine completely protects mice against lethal challenge of H5N1

Highly pathogenic avian H5N1 viruses may give rise to the next influenza pandemic due to their reassortment and mutation of the genome. Vaccine against this virus is important for coping with its potential threat. Chimpanzee adenovirus (Ad) vectors are a novel type of vaccine vectors that share the advantages of human serotype Ad vectors but without being affected by pre-existing human neutralizing antibody to the vaccine vector. Based on a replication-deficient chimpanzee Ad vector, AdC7, we generated a novel H5N1 vaccine candidate AdC7-H5HA that expresses H5N1 Hemagglutinin (HA). When tested in mice, the vaccine significantly reduced the virus load and pathological lesions in the lung tissues, and conferred complete protection against lethal challenge by a homologous virus. Mechanistically, the AdC7-H5HA vaccine can induce both HA-specific humoral and cell-mediated immune responses in mice. Also, sera transfer experiments demonstrated that neutralizing antibodies alone could provide protection. In conclusion, our results show that chimpanzee Ad vector expressing influenza virus HA may represent a promising vaccine candidate for H5N1 viruses and other influenza virus subtypes.     

AcSDKP经由PDGF介导的细胞外信号调节激酶1/2的调节通路在大鼠矽肺纤维化形成中的作用

探讨抗纤维化短肽N-乙酰基-丝氨酰-天门冬酰-赖氨酰-脯氨酸(AcSDKP)经由血小板源性生长因子(PDGF)介导的细胞外信号调节激酶1/2(ERK1/2)的调节通路在矽肺纤维化形成中的作用。采用一次性支气管内灌注二氧化硅(SiO2)粉尘制作矽肺大鼠模型,将其分为对照组(4周组和8周组)、矽肺模型组(4周和8周组)、AcSDKP治疗组(抗纤维化治疗组和预防治疗组)。采用贴块法进行肺成纤维细胞的原代和传代培养。HE染色观察肺组织形态学变化,Western blot法检测肺组织、肺成纤维组织Ⅰ型、Ⅲ型胶原蛋白和ERK1/2及磷酸化-ERK1/2蛋白的表达以及肺组织PDGF及其受体蛋白的表达。与对照比较,矽肺大鼠肺内PDGF及其受体蛋白、Ⅰ型和Ⅲ型胶原蛋白以及磷酸化-ERK1/2蛋白表达均增强,而AcSDKP治疗组则见上述蛋白表达减弱;在培养的肺成纤维细胞,PDGF能够刺激其Ⅰ型、Ⅲ型胶原蛋白表达增加,同时上调磷酸化-ERK1/2蛋白的表达,而AcSDKP则显示出与PD98059(细胞外信号调节激酶通路特异性抑制剂)相同的作用,即能够抑制PDGF刺激成纤维细胞Ⅰ型、Ⅲ型胶原蛋白表达和上调磷酸化-ERK1/2蛋白表达...     

Safety and immunogenicity of inactivated,Vero cell culture-derived whole virus influenza A/H5N1 vaccine given alone or with aluminum hydroxide adjuvant in healthy adults

Dosage-sparing strategies, adjuvants and alternative substrates for vaccine production are being explored for influenza vaccine development. We assessed the safety and immunogenicity of a Vero cell culture-grown inactivated whole virus influenza A/H5N1 vaccine with or without aluminum hydroxide adjuvant [Al(OH)₃] in healthy young adults. Vaccines were well tolerated, but injection site discomfort was more frequent in groups receiving Al(OH)₃. Dose-related increases in serum antibody levels were observed. Neutralizing antibody titers varied significantly when tested by two different laboratories.     

P13K、P38 MAPK及ERK1/2抑制剂对EGF诱导的血管平滑肌细胞迁移的实验研究

目的 探讨P13K抑制剂Wortmannin、P38 MAPK抑制剂SB202190及ERK1/2抑制剂PD98059对表皮生长因子(EGF)诱导的大鼠主动脉平滑肌细胞(VSMCs)迁移的影响.方法 以1%FCS血清孵育为对照,用EGF(10ng/ml)刺激,平行加入PD98059(PD组)、SB202190(SB组)和Wortmannin(WT),划痕损伤实验分析VSMCs迁移情况.结果 划痕后24 h,EGF组VSMCs迁移较对照组明显(P<0.01),WT组、SB组及PD组与EGF组相比都明显抑制VSMCs迁移(P<0.01),但三个抑制剂组之间差异无统计学意义(P>0.05);划痕后30 h,EGF组VSMCs迁移仍较对照组明显(P<0.01),WT组、SB组及PD组与EGF组相比则明显抑制VSMCs迁移(P<0.01),且三个抑制剂组之间差异有统计学意义,其中SB组较WT组和PD组抑制VSMCs的迁移更明显(P<0.01).结论 EGF诱导VSMCs迁移可能通过P13K、P38 MAPK及ERK1/2途径起作用,且P38 MAPK途径作用更明显.     

牛磺酸对视网膜感光细胞光损伤及NFkB/caspase-1表达的影响

目的:观察牛磺酸对光化学损伤后感光细胞凋亡的影响,并探讨其防护机制。方法:70只SD大鼠随机分为对照组(control),牛磺酸组(taurine,Tau),分别喂饲标准饲料或添加4%Tau饲料15d后接受3000±200lx持续0、1、3、6、9、12、24h光照,用TUNEL法检测感光细胞凋亡并计算凋亡指数(apoptoticindex,AI),免疫印迹法测定caspase-1和核内p65蛋白表达水平,RT-PCR法检测IκBamRNA。结果:光照9h后Tau组出现凋亡细胞,且多个时相点该组AI值显著低于对照(P<0.05);短期光照(3h/6h)诱导p65核转位,Tau组核内p65量显著高于对照组(P<0.05),长时程光照后(12h/24h)对照组核内p65显著降低,Tau组仍有较高表达;光照1h后对照组IκBamRNA迅速升高,而Tau组在光照3、6h后升高较显著。视网膜caspase-1蛋白随光照时间延长表达增高,Tau组显著低于对照组对应时相点(P<0.05)。结论:牛磺酸抑制视网膜光化学损伤中感光细胞凋亡,可能与其维持NFκB激活状态进而下调caspase-1表达有关。     

碱性成纤维细胞生长因子对卵巢癌的影响

目的观察Ras-Raf-ERK 1/2途径介导的碱性成纤维细胞生长因子(bFGF)对卵巢癌细胞系CAOV3细胞增殖和凋亡影响,探讨bFGF及其信号转导途径与卵巢癌发生发展关系。方法以bFGF和促细胞分裂剂激活性蛋白激酶1(MEK1)抑制剂PD98059处理CAOV3细胞,用四甲基偶氮噻唑蓝(MTT)法检测细胞增殖情况,流式细胞术检测细胞凋亡情况,蛋白印迹(Western blot)检测细胞外信号调节蛋白激酶1/2(ERK1/2)的活性。结果bFGF处理后细胞增殖比明显增加,且与bFGF水平呈剂量依赖关系,bFGF浓度为75 ng/ml时细胞增殖比最高为140%;bFGF使无血清诱导的凋亡细胞比例下降[(33.20±5.32)%~(2.38±3.36)%];bFGF诱导ERK1/2活性增高。PD98059可抑制bFGF的这些作用。结论bFGF通过Ras-Raf-ERK 1/2途径介导,促进卵巢癌CAOV3细胞增殖,抵抗无血清诱导凋亡。在卵巢癌发生发展过程中,bFGF信号传递发挥了重要作用。     

流感病毒国际代表株B/山东/07/97的抗原特点分析

[目的]对B型流感病毒国际代表株B/山东/07/97进行抗原特点分析并与流行株相比较,从而判断该株今后可能引起的流行情况及疫苗的保护效果.[方法]用流感病毒血凝抑制试验检测流感病毒血凝素的抗原性,对血凝素重链区(HA1)进行基因序列分析.[结果]与在国际上流行的两组抗原系的毒株相比较,B/山东/07/97具有B/Victoria/2/87系毒株的特点,与原代表株比较具有变异性,与1998～1999流行季节新分离株比较具有代表性,并且与B/Yamagata/16/88系毒株的抗原性差异相对较小.[结论]B/山东/07/97类毒株1999～2000年可能会在更大范围发生流行;含该毒株的疫苗对两个抗原系的B型流感病毒感染均能产生良好的保护效果.     

甲型流感病毒NS1蛋白应用研究进展

流感病毒是引起急性流感的病原体,具有传染性强、变异快和宿主范围广的特点.随着对流感病毒研究的深入,人们对流感病毒的认知度不断提高,对其的开发利用也随之启动,其中对最短的NS基因及其编码蛋白(NS1和NS2)的研究开发更令人注目.NS1蛋白虽在病毒出芽时未被包装,但在感染细胞中发挥了多种生物功能,如对宿主细胞IFN的调节...     

高致病性禽流感H5N1病毒M1蛋白的重组表达及在感染检测中的初步应用

目的:原核表达重组高致病性禽流感基质蛋白M1并分析其在感染检测中的作用。方法:根据NCBI公布的基质蛋白M1的核苷酸序列设计特异性引物,用高致病禽流感H5N1病人分离的病毒提取病毒RNA进行反转录合成cD-NA,扩增基质蛋白M1基因。将M1基因克隆到pQE30原核表达载体进行非融合表达,纯化重组表达的蛋白并用免疫学方法检测分析。结果:克隆了高致病禽流感H5N1病毒基质蛋白M1基因,并采用pQE30原核表达载体成功表达该蛋白。以该重组蛋白为检测包被抗原建立间接ELISA,检测6份病人血清标本均为阳性,而正常对照人群为阴性。结论:高致病禽流感H5N1病毒M1蛋白可以应用于感染检测中。     

几种甲型H1N1流感病毒SYBRGreenI荧光RT—PCR检测方法的验证

目的比较和验证5种甲型H1N1流感病毒SYBRGreenI荧光RT—PCR检测方法。方法首先使用美国NCBI网站的Primer—BLAST程序验证5种甲型H1N1流感病毒SYBRGreenI荧光RT—PCR检测方法引物的理论特异性,然后使用猪流感H1N1病毒核酸、2009年新甲型H1N1流感病毒核酸、H5N1禽流感核酸、能力验证样品作为试验样品验证方法的实验特异性。结果理论验证结果显示5对来源于文献的引物均适合于猪流感的检测,前3对可用于新型甲型H1Nl流感的检测,但只限于个别来源的毒株,与预期略有差别。实验验证结果显示,方法l可正确检出甲型流感病毒;方法2可正确检出猪流感H1N1亚型病毒、新甲型H1N1流感病毒核酸;方法3检测新甲型H1N1流感核酸时未获得阳性结果;方法4可以正确检测出猪流感H1病毒核酸;方法5可以正确检测出猪流感H1N1病毒核酸,但检测新甲型H1N1流感核酸、禽流感H5N1核酸时也为阳性。结论方法1可用于猪甲型流感和新甲型H1N1流感病毒的初筛;方法2可用于猪流感H1N1亚型病毒和新甲型H1N1流感病毒核酸的初筛;方法3不适合新甲型H1N1流感病毒核酸的检测;方法4可用于猪流感H1亚型的检测;方法5不适合于猪流感H1N1亚型病毒的检测。采用SYBRGreenI荧光RT—PCR方法检测甲型H1N1流感病毒核酸,特异性普遍不十分理想,只能用于初筛检测。     

2009年深圳新甲型H1N1流感病毒分离鉴定及分离株特征分析

目的比较新甲型H1N1流感病毒对MDCK细胞和鸡胚的敏感性差异,了解病毒分离株特征,为疫苗制备和开展实验室常规监测等奠定基础。方法用MDCK细胞和鸡胚进行流感病毒分离[1],收获病毒后提取病毒RNA,逆转录-聚合酶链式反应(RT-PCR)扩增,阳性扩增产物纯化后测序,测序结果使用Clustal和MEGA4.1软件进行比较分析,通过与NCBI数据库中不同时间和地区的流感病毒H1N1株同源比对后,绘制种系发生树。结果从334份患者呼吸道标本中,用MDCK细胞分离法分离出134株新甲型H1N1病毒,分离率为40.12%,用鸡胚分离法分离出150株新甲型H1N1病毒,分离率为44.91%;所测10株新甲型H1N1毒株HA1区的344个氨基酸,与2009年新甲型H1N1国际代表株A/California/07/2009相比,只有少数位点发生氨基酸替换,并且这些位点都不在抗原决定簇上;种系发生树显示,10株新甲型H1N1和国际代表株同源性较大,形成一簇,与A/Swine/guangxi/13/2006较为接近。结论新甲型H1N1病毒对MDCK细胞和鸡胚的敏感性相差不大;新甲型H1N1病毒未发生变异,短期内不会产生新变种。     

甲型H1N1病毒HA、NA氨基酸序列比较及抗原性表位预测

目的：比较新型甲型H1N1流感病毒分离株HA、NA氨基酸序列之间的差异,分析HA、NA蛋白可能的抗原性细胞表位。方法：从GenBank中选取28株世界各地的新型甲型H1N1流感病毒分离株并下载各株HA、NA的氨基酸序列;使用ClustalX和MEGA2.0软件对氨基酸序列的进行进化树分析;用Protean软件预测HA、NA蛋白的B细胞表位;用NetMHCⅡ2.2预测T细胞抗原表位。结果：世界范围的毒株之间氨基酸序列相对保守,HA及NA均只有少数位点的变异,HA蛋白氨基酸序列之间较NA蛋白氨基酸序列之间变异较大;预测HA蛋白具有8个B细胞表位和10个T细胞表位;NA蛋白有12个B细胞表位和7个T细胞表位。结论：HA、NA蛋白的少数区域抗原性相对比较稳定,可以作为检测以及疫苗研究的靶区域。