BCG感染树突状细胞的转录组测序和分析

目的 通过转录组学筛选DC2.4细胞内BCG感染相关的差异表达基因和信号通路,为探索DC细胞抗分枝杆菌的胞内免疫机制提供科学依据。方法 以小鼠DC2.4细胞作为细胞模型,设置感染组和未感染组,感染组经BCG(MOI=2)感染,培养24 h后收集细胞,采用转录组测序技术(RNA-Seq)检测感染组和未感染组mRNA表达谱并结合生物信息学分析方法,筛选树突状细胞内与BCG感染相关的差异表达基因并进行qRT-PCR验证。结果 与未感染组相比,感染组差异表达基因共有27个(P<0.05),其中26个上调基因,1个下调基因。通过GO和KEGG富集分析发现差异基因参与炎症反应、免疫系统过程等;TNF信号通路、IL-17信号通路以及细胞因子与细胞因子受体信号通路等参与BCG胞内感染过程。qRT-PCR验证与BCG感染相关的差异表达基因与转录组数据趋势一致。结论 BCG感染树突状细胞后,能够激发宿主细胞炎症反应和免疫应答,TNF、IL-17等信号通路参与BCG胞内感染过程。     

弓形虫ROP16Ⅱ效应分子对宿主A549细胞基因表达谱的影响

目的 ROPl6在弓形虫入侵宿主细胞的过程中发挥着关键作用。为了提高ROP16对宿主细胞的分子功能的认识,本研究分析了ROP16_Ⅱ基因转染的A549细胞株的表达谱,筛选了新的候选基因,为揭示Ⅱ型弓形虫病的发病机制提供了依据。方法 通过表达谱芯片技术对过表达ROP16_Ⅱ的A549细胞株的表达谱进行研究,并从中筛选差异表达基因。利用ToppGene从差异表达基因中筛选候选基因,并通过RT-qPCR对获得的基因进行验证。结果 共获得了9 994个差异表达基因,其中上调的基因有4 293个,下调的基因有5 701个。将这些差异表达基因进行 GO 功能富集分类,主要涉及多种生物学过程,包括发育过程、生物调控、免疫应答和信号传导等过程。采用ToppGene筛选得到4个疾病候选基因,其中上调表达的有IL4R和STAT1;下调表达的有IL12RB1和IL2RG。RT-qPCR结果与表达谱芯片数据一致。结论 ROP16_Ⅱ入侵宿主细胞后影响宿主细胞基因表达谱,了解ROP16_Ⅱ的作用机制,对于更加全面地揭示Ⅱ型弓形虫病的发病机制具有重要意义。     

基于生物信息学探讨弓形虫ROP16Ⅰ对A549细胞表达谱的影响

目的 采用生物信息学对ROP16_Ⅰ基因转染的A549细胞株的表达谱芯片结果进行数据挖掘,寻找影响Ι型弓形虫疾病的差异表达基因,为揭示Ι型弓形虫疾病的发病机制提供依据。方法 通过生物信息学方法对过表达ROP16_Ⅰ的A549细胞株的表达谱进行研究,从中筛选差异表达基因。将得到的数据导入在线分析工具ToppGene和STRING中对差异表达基因进行筛选,同时进行GO 分析及KEGG分析。结果 本研究共获得了483个差异表达基因,其中上调的基因共有252个,下调的基因共有231个。ToppGene最终筛选得到CCL8、CXCL11、PIAS1及EIF2AK2等14个Ι型弓形虫相关的基因。GO分析和KEGG分析发现,这些候选基因与白介素IL-10、IL-4、IL-13信号通路,细胞因子-细胞因子受体相互作用通路及Toll样受体信号通路等相关。在蛋白-蛋白互作网络中,这14个候选基因恰好处于网络的最核心位置。结论 Ι型弓形虫疾病的发生可能与CCL8、CXCL11、PIAS1及EIF2AK2等基因密切相关。     

基于GEO数据分析参与MERS-CoV感染致病的信号传导通路和关键分子

目的 基于基因表达谱芯片数据,筛选和分析中东呼吸综合症病毒(Middle East respiratory syndrome coronavirus, MERS-CoV)感染人类呼吸道上皮细胞后的差异基因,确定参与致病的信号传导通路和关键分子。方法 在美国国立生物技术信息中心的Gene Expression Omnibus(GEO)数据库搜选MERS-CoV病毒感染支气管上皮细胞表达谱数据,使用在线分析工具GEOR分析MERS-CoV感染支气管上皮细胞正常组和感染组各种基因的表达情况,以具有统计学意义的在表达量上升或下降2倍以上的差异基因为研究对象,使用基因功能分析注释工具DAVID对差异基因进行基因本体分析和信号传导通路分析,使用STRING构建基因间相互作用网络,利用cytoscape筛选相互作用网络的关键基因。结果 对比感染与未感染组的基因表达,筛选到了差异基因1 553个,其中上调基因850个,下调基因703个。基因功能富集分析结果提示这些差异基因涉及免疫反应,炎症反应,凋亡过程,支气管纤毛形成和运动等多条信号传导通路。结论 研究MERS-CoV感染肺部支气管细胞基因表达谱,发现在细胞中起重要作用的多条信号通路的异常,筛选到多个关键基因,这些信号通路可能在病毒致病过程中起到重要作用。本研究将为揭示MERS-CoV 致病的分子机制提供帮助,为确定新的治疗靶标和策略提供数据。     

鸭源H7N9亚型禽流感病毒感染SPF鸡转录组学分析

目的 为了分析鸭源H7N9亚型禽流感病毒感染SPF鸡后宿主基因表达水平的变化。方法 以鸭源H7N9亚型禽流感病毒感染SPF鸡,收集肺脏进行高通量测序。结果 与对照组相比,感染组得到差异表达基因740个,其中上调基因有602个,下调基因有138个。GO条目分析发现,差异基因主要涉及免疫应答反应和炎症反应等。经KEGG 数据库比对注释及富集分析显示有7个通路富集显著,其中Toll-like信号通路有11个基因表达上调,分别为IL-6、TLR4、PIK3、IRF7、MD-2、IRF5、MYD88、CD86、STAT1、TLR2和CCL4,NOD-like受体信号通路有7个基因表达上调,分别为IRF7、CTSB、P2RX7、CYBB、PSTPIP1、HSP90AA1和NAMPT。结论 鸭源H7N9亚型病毒感染SPF鸡后,免疫相关基因表达明显增强。在Toll-like信号通路中, TLR4在MD-2的协助下被激活,随后依赖MYD88途径激活下游的IRF5,继而引起CCL4、IL-6显著表达。同时NLRP3炎症体在H7N9亚型病毒感染过程中也发挥着重要作用。     

转录组分析布鲁氏杆菌患者外周血免疫基因的表达情况

目的 通过生物信息学分析方法探讨布鲁氏杆菌病的关键失调免疫基因和分子机制。方法 从GSE69597中获取布鲁氏杆菌病患者全血转录组表达数据。通过差异分析筛选布鲁氏杆菌病患者和对照之间的差异表达基因(DEGs),并利用immport数据库调取DEGs中免疫相关的基因集(immune-DEGs)。通过Enirchr在线富集工具对immune-DEGs进行富集分析。构建immune-DEGs的蛋白质-蛋白质互作(PPI)网络,并鉴定网络中的高度互联的核心(hub)基因。利用qRT-PCR验证hub基因的表达,并绘制hub基因的ROC曲线。使用ssGSEA算法评估布鲁氏杆菌病患者中免疫细胞的评分,并通过流式细胞术检测血液样本中免疫细胞的水平。结果 共获得390个immune-DEGs,富集结果中发现了T细胞受体信号通路和Th17细胞分化等。在10个hub基因中IFNG和TNF在布鲁氏杆菌组中显著高表达。ROC曲线表明IFNG对布鲁氏杆菌病具有良好诊断意义。此外,活化型CD₄ T细胞、效应型CD₄ T细胞、效应记忆型CD₈ T细胞和2型T辅助细胞因子在布鲁氏杆菌患者中明显增多。流式细胞术检测发现与健康对照组相比,布鲁氏杆菌病患者外周血中Th2和Th17细胞比例增高,Th1和Treg细胞比例则降低。结论 本研究结果不仅提高了我们对布鲁氏杆菌感染后机体免疫反应的认识,还为诊断和治疗布鲁氏杆菌病提供了更多的方向。     

微小隐孢子虫IId亚型早期感染对HCT-8细胞TLRs的影响

目的 研究微小隐孢子虫IId亚型(中国流行株)感染对HCT-8细胞TLRs的影响。方法 微小隐孢子虫IId亚型感染HCT-8细胞4 h及12 h后,提取细胞RNA,利用qRT-PCR方法检测TLRs的表达水平,并通过生物信息学分析表达差异显著的miRNAs与TLRs的关系。结果 HCT-8细胞表达TLR1-TLR10 10种TLRs,并且微小隐孢子虫IId亚型早期感染(4 h和12 h)导致TLR2和TLR4表达量较对照组上调有统计学意义,其它8种TLRs较对照组表达变化无统计学意义。生物信息学分析表明TLR2和TLR4 并不是表达差异显著的miRNAs靶基因,但这些表达差异显著的miRNAs可以靶向27个TLRs信号通路基因。结论 TLR2和TLR4在微小隐孢子虫IId亚型感染HCT-8细胞中可能发挥着一定的作用,差异表达miRNAs的预测靶向中有TLR信号通路相关基因。     

综合基因表达谱分析与人类肝胰岛素抵抗相关蛋白分子表达研究*

目的 分析糖尿病患者与正常人肝组织之间的差异表达基因（DEGs）,筛选出肝组织中与可能产生胰岛素抵抗相关作用的蛋白分子。方法 从GEO数据库下载基因芯片数据集GSE23343（包括10例2型糖尿病和7例正常人肝组织的基因表达值）,通过DEGs表达谱分析和功能通路富集分析,构建DEGs对应的蛋白质-蛋白质相互作用网络。结果 分析得到928个显著上调的DEGs（P<0.01）,发现DEGs主要富集在细胞和代谢生物过程中,KEGG通路富集显示DEGs主要集中于信号转导和肿瘤相关通路。经蛋白质相互作用网络构建,筛选出5个关键蛋白分子MDM2、PCNA、CAV1、PIK3R1、NR3C1。结论 系统地筛选出人类肝组织中可能与胰岛素抵抗形成相关的蛋白分子,为进一步实验研究肝胰岛素抵抗产生机制和新的降血糖药物作用靶点提供基础。     

戊型肝炎病毒与ING5相互作用的研究

目的 构建ING5基因真核表达载体和ING5荧光素酶载体,将pMIR-Report-ING5荧光素酶质粒与PUC-HEV质粒共转染293T细胞后,验证HEV与ING5之间相互关系。方法 通过提取293T细胞总RNA,RT-PCR法扩增ING5基因序列,并克隆到真核表达载体PGC3.1 (+)上。利用脂质体转染法将PGC-ING5真核表达质粒转染HepG2细胞后,通过 Western Blot法检测ING5表达情况;将pMIR-Report-ING5荧光素酶质粒和PUC-HEV质粒共转染293T细胞进一步探究ING5与HEV之间相互作用关系。结果 经双酶切和测序鉴定真核表达质粒PGC-ING5构建成功,ING5基因在HepG2细胞中成功表达;将pMIR-Report-ING5荧光素酶质粒与PUC-HEV质粒共转染293T细胞后,发现HEV明显抑制239T细胞中pMIR-Report-ING5荧光素酶的活性。结论 成功构建了PGC-ING5真核表达载体和pMIR-Report-ING5荧光素酶载体,并且证明HEV与ING5之间具有相互作用。     

人工诱变利福平抗性布鲁氏菌转录组测序分析

目的 筛选布鲁氏菌中参与利福平代谢相关基因(除rpoB基因外)。方法 利用人工诱变技术获得利福平抗性菌株,通过转录组测序获得标准菌株和利福平抗性菌株全基因组水平的基因表达量,利用EBSeq算法筛选差异表达基因,预测利福平代谢相关基因及主要代谢途径。结果 通过不同浓度的利福平人工诱变,能够获得抗性表型稳定的布鲁氏菌变异菌株。转录组测序发现利福平抗性菌株中有121个基因表达量上调,197个基因表达量下调,差异基因功能主要集中于催化活性、细胞膜和细胞成分、代谢过程和细胞过程;主要参与抗生素的生物合成、细菌分泌系统和ABC 转运蛋白等代谢通路。结论 包括virB操纵子在内的涉及碳代谢等代谢通路的基因,通过表达量的改变参与抵抗利福平的作用。 本研究为布鲁氏菌耐药相关基因的筛选提供新思路,同时为布鲁氏菌耐药菌株的防控提供基础性数据。     

冠状病毒感染相关心肌损伤机制的组学分析及治疗药物的预测

目的研究冠状病毒感染相关心肌损伤机制并预测可能有效的治疗药物。方法在基因表达数据库检索并筛选得到GSE59185数据集,根据不同的亚型分为wt组、ΔE组、Δ3组、Δ5组和对照组。用R语言Limma程序包对各组进行差异表达基因分析,将各组上调、下调表达基因分别取交集,作为共同差异表达基因,在DAVID数据库进行基因本体学(GO)和京都基因与基因组百科全书(KEGG)通路富集分析。采用自主研发的表观精准治疗预测平台(EpiMed)进行治疗药物预测。用STRING数据库对共同差异表达基因构建蛋白质互作网络并筛选核心基因。结果各组差异表达基因分析,共交集上调基因191个,下调基因18个,共同差异表达基因共209个。GO富集分析发现,共同差异基因主要富集在病毒反应、病毒防御反应、Ⅰ型干扰素反应、γ干扰素调节、γ干扰素介导的信号通路、先天免疫反应调节等;KEGG通路富集主要与细胞因子与受体相互作用、病毒蛋白与细胞因子和细胞因子受体相互作用、TNF、Toll样受体、缺氧诱导因子1及白细胞介素17信号通路等有关。通过EpiMed预测的药物主要为白藜芦醇、利托那韦、维甲酸、连翘、鱼腥草等。网络分析筛选得到干扰素调节因子7、干扰素刺激基因15、抗粘液病毒基因1、β型蛋白酶体亚基8、干扰素调节因子9、 2′,5′-寡聚腺苷酸合成酶(OAS)1、OAS2、OAS3、含基本S腺苷蛋氨酸域2、2′,5′-寡聚腺苷酸合成酶样等核心基因。结论多个炎症通路的异常活化可能是冠状病毒感染后患者发生心肌损伤的原因。白藜芦醇、利托那韦、维甲酸、连翘、鱼腥草可能对此具有治疗作用。     

Clinical course and duration of viremia in vertically transmitted hepatitis E virus (HEV) infection in babies born to HEV-infected mothers

Summary.  Infection with the hepatitis E virus (HEV) causes a self-limiting acute hepatitis. However, prolonged viremia and chronic hepatitis has been reported in organ transplant recipients. Vertically transmitted HEV infection is known to cause acute hepatitis in newborn babies. The clinical course and duration of viremia in vertically transmitted HEV infection in neonates in not known. We studied 19 babies born to HEV infected mothers. Babies were studied at birth and on a monthly basis to evaluate clinical profile, pattern of antibody response and duration of viremia in those infected with HEV. Fifteen (78.9%) babies had evidence of vertically transmitted HEV infection at birth (IgM anti-HEV positive in 12 and HEV RNA reactive in 10) and three had short-lasting IgG anti-HEV positivity because of trans-placental antibody transmission. Seven HEV-infected babies had icteric hepatitis, five had anicteric hepatitis and three had high serum bilirubin with normal liver enzymes. Seven babies died in first week of birth (prematurity 1, icteric HEV 3, anicteric HEV 2 and hyperbilirubinemia 1). Nine babies survived and were followed up for clinical, biochemical, serological course and duration of viremia. Five of 9 babies who survived were HEV RNA positive. HEV RNA was not detectable by 4 weeks of birth in three babies, by 8 weeks in one and by 32 weeks in one. All surviving babies had self-limiting disease and none had prolonged viremia. Thus HEV infection is commonly transmitted from mother-to-foetus and causes high neonatal mortality. HEV infection in survivors is self-limiting with short lasting viremia.     

病毒感染时IFITM3与TRIM22和Foxp3表达及关联基因的富集分析

目的病毒感染时对IFITM3、TRIM22和FOXP3及基因进行富集分析。方法利用STRING作出IFITM3、TRIM22和FOXP3及其关联的50个基因互作网络图;利用DAVID数据库和KOBAS数据库对其进行GO分析和KEGG分析。应用荧光定量PCR对3基因的mRNA相对表达量进行相关性分析。结果通过STRING数据库分析得到3基因互作网络图,关联性大小与图中代表基因节点的圆圈大小呈正比(P<0.05)。经基因功能富集分析表明基因相关的信号通路与基因有关联的可信度较高(P<0.05)。在乙型肝炎病毒、丙型肝炎病毒和甲型流感病毒感染患者的外周血白细胞(P<0.05)分别为(4.462±3.441)、(4.406±4.415)和(4.013±0.081)中的IFITM3的mRNA与健康对照组差异有统计学意义(t=2.987,P<0.01)。结论机体感染病毒后,IFITM3与TRIM22之间、FOXP3与TRIM22之间可能存在着一定的相互作用,并参与机体对病毒感染的应答进程。     

Disparity of basal and therapeutically activated interferon signalling in constraining hepatitis E virus infection

Hepatitis E virus (HEV) represents one of the foremost causes of acute hepatitis globally. Although there is no proven medication for hepatitis E, pegylated interferon‐α (IFN‐α) has been used as off‐label drug for treating HEV. However, the efficacy and molecular mechanisms of how IFN signalling interacts with HEV remain undefined. As IFN‐α has been approved for treating chronic hepatitis C (HCV) for decades and the role of interferon signalling has been well studied in HCV infection, this study aimed to comprehensively investigate virus–host interactions in HEV infection with focusing on the IFN signalling, in comparison with HCV infection. A comprehensive screen of human cytokines and chemokines revealed that IFN‐α was the sole humoral factor inhibiting HEV replication. IFN‐α treatment exerted a rapid and potent antiviral activity against HCV, whereas it had moderate and delayed anti‐HEV effects in vitro and in patients. Surprisingly, blocking the basal IFN pathway by inhibiting JAK1 to phosphorylate STAT1 has resulted in drastic facilitation of HEV, but not HCV infection. Gene silencing of the key components of JAK‐STAT cascade of the IFN signalling, including JAK1, STAT1 and interferon regulatory factor 9 (IRF9), stimulated HEV infection. In conclusion, compared to HCV, HEV is less sensitive to IFN treatment. In contrast, the basal IFN cascade could effectively restrict HEV infection. This bears significant implications in management of HEV patients and future therapeutic development.     

Expression profiles of host immune response‐related genes against HEV genotype 3 and genotype 1 infections in rhesus macaques

Hepatitis E virus (HEV) genotype (gt) 3 infection is food‐borne causing sporadic infections in older individuals and gt1 infection is waterborne, often causing epidemics affecting primarily young adults. Although HEV infection causes self‐limited disease, gt3 induces chronic infection in immunocompromised individuals. Hepatic host gene expression against gt3 infection remains unknown. Host gene expression profiles for HEV gt1 (n = 3) and gt3 (n = 7) infections were analysed in the livers of experimentally infected rhesus macaques. HEV RNA was detected from 2 to 24 days after inoculation (DAI) in stool and serum, elevated alanine aminotransferase (ALT) activity was detected from 7 to 31 DAI, and anti‐HEV antibody became detectable between 12 and 42 DAI. All 10 animals cleared the infection between 34 and 68 DAI. We found that 24%, 48% and 41% of hepatic immune response genes against gt3 infection were upregulated during the early, peak and decline phases of HEV RNA replication. For gt1 infection, 25% of hepatic immune response‐related genes were downregulated during early viremia, but 6%, 34% and 37% of genes were upregulated at the early, peak and during decline of HEV RNA replication, respectively. Our study demonstrated distinct differences in the expression profiles of host immune response‐related genes of HEV gt3 and gt1 infections in experimentally infected rhesus macaques.     

Hepatitis E: an overview

Hepatitis E is an enterically transmitted, acute, self-limited, icteric viral disease that occurs in large numbers in countries of the Indian subcontinent, Asia, and Africa. The frequency of epidemics and the high mortality rate among infected pregnant women are strong indicators that hepatitis E is an important cause of morbidity and mortality in humans. Several isolates of hepatitis E virus (HEV) derived from infected humans and experimental animals have recently been cloned and sequenced, allowing investigators to determine the molecular structure of the HEV genome. Laboratory diagnosis of HEV infection is done by detection of HEV antibodies, HEV RNA in stool and serum samples, HEV particles in stool specimens, and HEV antigen in hepatocytes and stool specimens. The detection of anti-HEV by enzyme immunoassay, with the use of several recombinant HEV proteins or synthetic peptides, is the most frequently applied method for the diagnosis of the infection and characterization of its epidemiologic features. Laboratory determination of HEV replication, immune response, and liver pathologic features in patients with hepatitis E and in infected primates has facilitated studies of the disease. Preventive measures against HEV infection include the passive transfer of protective antibodies or active immunization. In efforts to develop HEV vaccines, various recombinant proteins have been used. Although a range of protective immune responses have been induced in primates, further modifications of immunogen, adjuvant, and immunization schedules are necessary to prevent HEV infection. Much remains to be learned about epidemiology of HEV infection, reservoir(s) of the virus, and protective immunity in order to develop effective strategies to prevent hepatitis E.