致病性大肠埃希菌感染小鼠肾脏的基因表达谱分析

目的了解尿道致病性大肠埃希菌(UPEC)感染小鼠肾脏的基因表达谱差异变化,探究宿主早期免疫和炎症反应过程。方法 2013年8月分别利用UPEC132菌株和磷酸缓冲液(PBS)经尿道逆行感染小鼠各20只,感染后于4和8h处死并解剖取其肾脏,随机取两组中4和8h各2只小鼠单侧肾,提取其RNA,利用基因表达谱芯片筛选其差异表达基因,并利用京都基因与基因组百科全书(KEGG)进行信号通路分析。结果与PBS对照组相比,UPEC感染组小鼠肾脏在4和8h分别有204个和1 323个基因差异表达,KEGG通路分析共有59个差异表达基因直接与宿主细胞的6条炎症和免疫信号通路相关,包括细胞因子与其受体相互作用信号通路、抗原加工与呈递信号通路、丝裂原活化蛋白激酶(MAPK)信号通路、Toll样受体信号通路、白细胞跨内皮迁移信号通路及自然杀伤性细胞介导细胞毒作用信号通路。结论利用表达谱芯片筛选得到的59个差异表达基因成为研究宿主早期免疫防御UPEC感染的潜在目的基因,为进一步揭示宿主与UPEC相互作用机制奠定基础。     

金属的免疫学效应

环境中化学物质的免疫作用已成为近年来毒理学研究的敏感指标。一些实验证明,长期接触某些低浓度的金属及其化合物可能诱发宿主免疫应答的改变。主要表现为免疫抑制,包括宿主对感染原的易感性增强;吞噬细胞功能降低;免疫活性细胞及组织体液中的杀菌物质(溶茵酶、补体、干扰素等)的改     

免疫抑制BALB/c小鼠系统性白假丝酵母菌感染生物指标模型的建立

目的探讨经腹腔注射白假丝酵母菌建立免疫抑制BALB/c小鼠系统性感染的生物指标模型,为研究白假丝酵母菌感染的致病机制和抗真菌药物的药效学提供相关动物模型。方法对免疫抑制组BALB/c小鼠(腹腔注射环磷酰胺200 mg/kg·d,连续2 d)经腹腔注射白假丝酵母菌增强毒力株0.25 mL(浓度为1×10~(7 )CFU/mL)建立系统性白假丝酵母菌感染模型;取小鼠尾静脉血进行白细胞和中性粒细胞计数,取小鼠组织进行真菌镜检、培养、病理检查以及(1,3)-β-D-葡聚糖检测。结果免疫抑制组与对照组小鼠用药后第4天白细胞计数、中性粒细胞计数、平均体重比较,差异均具有统计学意义(均P0.05)。白假丝酵母菌感染组生存率为30.00%,对照组生存率为100.00%,两组生存率比较,差异有统计学意义(P0.05)。对注射真菌后第2~14天死亡小鼠以及第14天存活小鼠进行解剖,发现肺、肝、肾组织出现多处脓肿,以肾组织感染最为显著;小鼠组织真菌直接镜检可见大量菌丝体,组织培养均为白假丝酵母菌,组织病理可见大量菌丝体、炎细胞及组织坏死。白假丝酵母菌感染组肺、肾组织(1,3)-β-D-葡聚糖均增高,与对照组比较,差异均有统计学意义(均P0.05)。结论本实验方法可以成功建立免疫抑制BALB/c小鼠系统性白假丝酵母菌感染的动物模型。     

外阴阴道假丝酵母菌病免疫致病机制研究进展

外阴阴道假丝酵母菌病是一种常见的阴道黏膜真菌感染性疾病,对女性身心造成极大伤害.外阴阴道假丝酵母菌病反复发作而成复发性外阴阴道假丝酵母菌病.其发生、发展及转归与宿主的全身和局部免疫状态密切相关.随着研究深入,对假丝酵母菌的致病性及机体对阴道局部假丝酵母菌侵袭的反应,特别是机体和阴道局部对假丝酵母菌侵袭的免疫机制的研究有了一些新的认识.就近年外阴阴道假丝酵母菌病及复发性外阴阴道假丝酵母菌病的免疫致病机制研究进展做一综述.     

寡核苷酸表达谱揭示异烟肼耐药对结核分支杆菌致病性的影响

目的研究结核分支杆菌异烟肼耐药菌株与H37Rv异烟肼敏感菌株感染巨噬细胞后差异表达的基因. 方法利用高密度cDNA芯片检测巨噬细胞U937感染结核分支杆菌异烟肼耐药菌株与H37Rv后的基因表达谱的差异. 结果两者诱导巨噬细胞的差异表达基因有53条,异烟肼耐药菌株比敏感菌株诱导多种趋化因子和细胞免疫增强的细胞因子表达上调和免疫抑制细胞因子IL-10等表达下调. 结论异烟肼耐药对细菌毒力有明显的影响,异烟肼耐药菌株的致病力下降,异烟肼耐药菌株容易被宿主免疫系统清除;结果为耐多药结核病控制提供依据;骨桥接素和巨噬细胞趋化因子等,在抗异烟肼耐药结核分支杆菌感染免疫中的作用值得深入研究.     

克罗恩病人肠黏膜炎性区域真菌菌群组成变化研究

目的 :比较克罗恩病人肠黏膜炎性与非炎性区域真菌菌群组成的差异,探讨黏膜真菌菌群变化与克罗恩病的潜在联系。方法 :手术收集7例活动性克罗恩病人肠黏膜炎性与非炎性区域组织,提取DNA,采用巢式PCR扩增18S r RNA基因片段,行变性梯度凝胶电泳,确定肠黏膜真菌菌群多样性和组成。结果 :与非炎性黏膜相比,炎性区域真菌菌群丰富度和多样性显著升高(P0.05)。肠黏膜炎性区域真菌菌群组成发生显著改变,主要表现为条件致病白色念珠菌,热带假丝酵母菌,串珠状赤霉菌,甘蓝链格孢菌和新型隐球菌的相对丰度显著增加(P0.05);而共生菌酿酒酵母菌,芽殖酵母菌,产黄青霉菌和双色蜡蘑菌显著降低(P0.05)。结论 :克罗恩病人肠黏膜炎性区域真菌菌群严重失衡,条件致病真菌定植增加可能参与了肠黏膜炎性损伤的形成。     

真菌感染耐药率统计分析

近年来,随着大量抗生素、免疫抑制剂、皮质类固醇激素、抗肿瘤药物的应用,以及骨髓器官移植、艾滋病的出现使免疫抑制人群不断增多,导致正常菌群失调或抵抗力降低,致使由条件致病性真菌引起的侵袭性真菌感染日益增加。这些感染通常由条件致病性真菌引起,包括念珠菌、隐球菌、曲霉菌和毛霉菌等;这些真菌是宿主正常菌群的成员,正常情况下不致病,只有当宿主抵抗力降低时才引起疾病。随着侵袭性真菌感染患病率和病死率的不断增高,真菌感染的诊断和治疗备受关注,亟须临床实验室在真菌鉴定和药敏试验方面提供确实的辅助诊断,以便提高临床诊断和治疗水平。     

外阴阴道假丝酵母菌病免疫致病机制研究进展

外阴阴道假丝酵母菌病是一种常见的阴道黏膜真菌感染性疾病,对女性身心造成极大伤害。外阴阴道假丝酵母菌病反复发作而成复发性外阴阴道假丝酵母菌病。其发生、发展及转归与宿主的全身和局部免疫状态密切相关。随着研究深入,对假丝酵母菌的致病性及机体对阴道局部假丝酵母菌侵袭的反应,特别是机体和阴道局部对假丝酵母菌侵袭的免疫机制的研究有了一些新的认识。就近年外阴阴道假丝酵母菌病及复发性外阴阴道假丝酵母菌病的免疫致病机制研究进展做一综述。     

乙型肝炎病毒基因疫苗联合抗原蛋白免疫效应的评价

目的探讨乙型肝炎病毒(HBV)基因疫苗联合乙型肝炎病毒表面抗原S蛋白(HBsAg)免疫Balb/c小鼠的效应.方法构建编码S蛋白的重组真核表达质粒pCR3.1-S作为HBV基因疫苗.以HBV基因疫苗联合HBsAg蛋白免疫接种Balb/c小鼠,同时以HBV基因疫苗或S蛋白单独接种Balb/c小鼠作为对照.采用ELISA法检测免疫小鼠的抗HBs应答,3H-TdR掺入法检测免疫小鼠的淋巴细胞增殖反应.结果免疫接种2周、4周后,联合免疫组抗HBs滴度高于HBV基因疫苗单独接种组,但低于S蛋白单独接种组;6周后,HBV基因疫苗组抗HBs滴度最高,联合组次之.各组间免疫小鼠的淋巴细胞增殖反应无显著差异性(P＞0.05).结论 HBV基因疫苗联合S蛋白免疫Balb/c小鼠无优势.     

重组痘苗病毒嵌合巨蛋白的免疫学研究

目的　pSFJ1 6与pSFJ38均是以牛痘病毒包涵体 (ATI)启动子和分别串联的 1 6个和 38个p7 5突变型早期启动子为基本构件组成的复合型启动子 (ATI-p7 5)、痘苗病毒复制非必需区血凝素 (HA)基因为侧翼的痘苗病毒表达载体。分别插入编码艾滋病病毒 (HIV - 1 )外膜蛋白env、核心蛋白gag与编码干扰素 (IFNα - 2b)基因 ,观察其表达产物诱导的机体内产生抗体效价的变化规律。方法　利用分子克隆技术构建重组质粒 ,经脂质体转染与野生型痘苗病毒在Cos- 7细胞内同源重组 ,筛选、纯化、获得重组痘苗病毒vJ1 6env/IFNα - 2b和vJ38gag/IFNα - 2b。免疫小鼠后 ,经间接ELISA分折免疫小鼠血清抗体的OD4 90 值变化。结果　免疫小鼠后体内抗体的OD4 90 值 ,实验组与阴性对照组平均数有显著性差异 (P <0 0 5) ,实验组与阳性对照组之间无显著性差异 (P >0 0 5)。结论　重组痘苗病毒vJ1 6env/IFNα - 2b和vJ38gag/IFNα- 2b免疫小鼠后可诱导机体产生高滴度的抗HIV - 1env、gag与IFNα - 2b巨蛋白抗原的抗体 ,蛋白稀释度与血清抗体OD4 90 值呈负相关     

电磁脉冲诱导小鼠肠组织基因表达谱的变化

目的用基因芯片技术研究电磁脉冲(EMP)对小鼠肠组织的生物效应。方法将12只小鼠随机分为正常组和EMP组(每组6只),用200kV/m,200次的电磁脉冲辐照大鼠,于照后第18h,活杀小鼠,取空肠,提取RNA,经反转录后用Cy3、Cy5荧光标记,获得两组动物来源cDNA探针;cDNA探针与基因表达谱芯片进行杂交,结果由扫描仪扫描并用软件进行分析统计。结果EMP组与正常组相比,有56条基因表达发生了明显变化,其中有37条基因表达水平明显升高,而19条基因表达量明显下降。在表达异常的基因中有19条基因是已知功能或部分功能的基因,其中α-catenin胞质蛋白、淋巴细胞同型抗原、谷氨酰果糖6磷酸氨基转移酶、核糖体蛋白S17、小富脯氨酸蛋白2A、腺状激肽释放酶、脂氧合酶、醛酮还原酶、RNA结合蛋白、α-胰淀粉酶2、弹性蛋白酶2、p6-5淋巴细胞抑制因子基因等12条为表达上调基因;Jam新连接粘附分子、精氨酸甲基转移酶,Carm1、NNP-1、2-5寡腺苷酸合成酶、Mlark、ATP合成酶α亚基、解偶联蛋白基因等7条为表达下调基因,其余37条则为未知功能的基因。结论电磁脉冲辐照可诱导小鼠肠组织多个基因特异性表达,且上调基因数居多。     

Optimization of DNA vaccination against cutaneous leishmaniasis

The present studies were designed to examine the requirements of dose, route of inoculation and constituent antigens for the maintenance of complete and long lasting protection against cutaneous leishmaniasis due to Leishmania major conferred by a cocktail DNA vaccine encoding the Leishmania antigens LACK, LmST11 and TSA. Vaccination of C57Bl/6 mice with LACK DNA alone resulted in partial protection, whereas the combination of LmST11 and TSA provided stronger, though still incomplete protection compared to the combination of all three Ag DNAs. When intradermal (i.d), intramuscular (i.m.), and subcutaneous (s.c.) vaccination routes were compared, i.d. immunization reduced by five-fold the dose necessary to maintain complete protection. In vivo depletion of CD4+ or CD8+ T cells provided direct evidence that both populations are necessary to mediate complete protection. These results establish intradermal vaccination using DNA encoding multiple Leishmania antigens as a way to optimize priming of CD4+ and CD8+ T cells necessary for potent and durable protection against cutaneous leishmaniasis.     

用cDNA微列阵技术探讨苯中毒相关的DNA复制及损伤修复基因表达谱的改变

目的筛选与苯中毒有关的DNA复制及损伤修复基因。方法选取慢性苯中毒患者和无苯接触的健康人各7例配对，提取外周血白细胞总RNA。在反转录cDNA时，用Cy3，Cy5两种激发波长荧光分别标记两组样本cDNA探针。将各对探针混合后与含141条DNA复制及损伤修复的人类基因表达谱芯片杂交、扫描，分析杂交结果。结果共筛选出差异表达的基因25条，其中16条基因表达上调：9条基因表达下调，主要包括有DNA复制基因如PRIM2A，ORCIL等；DNA合成、重组及修复基因如POLK；DNA损伤信号基因／修复蛋白和DNA连接酶如RECQL，PRKDC，G22P1，ERCC3，ERCC1，CRY1。CHES1，BRCA2．APEX等；重组蛋白质如RECQL；其他DNA合成再重组和修复蛋白质如SKIV2L，RBMS1，SON．SET等。结论苯中毒患者外周血白细胞的DNA复制及损伤修复基因与正常人相比存在差异性表达，为进一步筛选苯中毒生物标志物提供了依据。     

Pro-apoptotic DNA vaccination ameliorates new onset of autoimmune diabetes in NOD mice and induces foxp3+ regulatory T cells in vitro

We have shown previously that incorporation of a cDNA coding for the pro-apoptotic protein BAX into plasmid DNA coding for a secreted form of the pancreatic beta-cell antigen glutamic acid decarboxylase (GAD) promotes prevention of type 1 diabetes in non-obese diabetic (NOD) mice. Here we present evidence indicating that injection of the same vaccine at time of early diabetes onset could ameliorate the disease with efficacy, with 42% of mice overtly diabetic by 40 weeks of age compared to 92% in control groups. In addition, immunological analysis revealed that the DNA vaccine induced CD4(+)CD25(+) T cells cultured from draining lymph nodes that had immunosuppressive function in vitro. The induced regulatory T cells (Tregs) expressed the foxp3 gene and showed cell-contact-dependent as well as TGF-beta- and IL-10-independent immunosuppressive activity. Data also revealed that CD4(+)CD25(-) T cells from mice immunized with the DNA vaccine yielded a cell population that was foxp3(+), showed increased expression of CD25 compared to control, and had immunosuppressive function in vitro, indicating that Tregs could have developed from antigen-induced, peripheral T lymphocytes. In contrast, injection of DNA coding for SGAD55 or BAX alone did not induce Tregs. Altogether, our data confirm that pro-apoptotic DNA vaccination can be used as an immunosuppressive strategy and demonstrate its potential for therapy of pathological autoimmunity.     

Protection of mice and guinea pigs against tuberculosis induced by immunization with a single Mycobacterium tuberculosis recombinant antigen, MTB41

MTB41 is a Mycobacterium antigen that is recognized by CD4+ T cells early after experimental infection of mice with Mycobacterium tuberculosis and by PBMC from healthy PPD positive individuals. Immunization of mice with plasmid DNA encoding the MTB41 gene sequence results in the development of antigen-specific CD4+ and CD8+ T cells, and protection against challenge with virulent M. tuberculosis. In the present studies, in contrast to DNA immunization, we show, that a strong MTB41-specific CD4+ T cell response, but no MHC class I restricted cytotoxic T lymphocyte (CTL) activity is detected in the spleen cells of infected mice. Therefore, this data suggests that the induction of CD8+ T cell response to MTB41 epitopes by DNA immunization may not be relevant to protection because these epitopes are not recognized during the infectious process. We also compared the repertoire of rMTB41 epitope recognition by CD4+ T cells of M. tuberculosis-infected mice with the recognition repertoire of mice immunized with the recombinant rMTB41 protein. Both regimens of sensitization lead to the recognition of the same molecular epitope. Coincidentally, immunization with the soluble recombinant protein plus adjuvant, a regimen known to generate primarily CD4+ T cells, resulted in induction of protection comparable to BCG in two well-established animal models of tuberculosis (mice and guinea pigs).     

国内临床分离阿萨希毛孢子菌3个新基因型分析

目的 通过对IGS1区基因序列分析,明确国内临床分离8株阿萨希毛孢子菌(T.asahii)的基因型,为T.asahii感染的流行病学研究提供依据.方法 以国内不同地区、不同患者临床分离的8株T.asahii为研究对象,PCR扩增、克隆IGS1区,寻找基因突变位点,构建系统发生树,明确国内T.asahii的基因型.结果 IGSI区长度均为643 bp,部分菌株碱基发生突变;8株T.asahii共发现5个基因型,其中3株为国际上首次发现的新基因型,分别命名为基因型10(BZ702)、11(BZ704)、12(BZ901),3株为基因型I(BZ701、BZ705、BZ706),2株为基因型4(BZ703、BZ902).结论 中国的T.asahii菌株具有不同于其他国家的多种基因型,种内多样性更丰富,这可能与中国地域广阔有关,为T.asahii感染的流行病学研究提供了依据.     

Allergen immunotherapy in intermittent allergic rhinitis reduces the intracellular expression of IL-4 by CD8+ T cells

BACKGROUND: T helper subset dysregulation is evident in allergic disorders. The role of T cytotoxic subsets is less understood. We investigated whether allergen immunotherapy in intermittent allergic rhinitis influences the intracellular expression of IL-4 and IFN-gamma by CD3+CD8(-) and CD3+CD8+ cells. METHODS: Nineteen adult patients with intermittent allergic rhinitis were evaluated before the pollen season, and then after one preseasonal course of subcutaneous allergen immunotherapy. Twelve healthy nonatopic patients matched for age and sex served as controls. Intracellular expression of IFN-gamma and IL-4 by CD3+CD8(-) (Th1 and Th2, respectively) and CD3+CD8+ (Tc1 and Tc2, respectively) was estimated by flow cytometry in peripheral blood cells after stimulation with PMA and ionomycin. RESULTS: Before immunotherapy the percentages of Th1, Th2, Tc1 and Tc2 did not significantly differ between the patients and the controls. After immunotherapy the percentage of Tc2 was lower in the rhinitic patients than in the controls (0.38% vs. 0.45%, p=0.04). The percentage of Tc2 cells decreased significantly after immunotherapy in the intermittent allergic rhinitis group (0.64% vs. 0.38%, p=0.02) with tendency to decrease in ratios of Tc2/Tc1 (p=0.059) and with no changes in ratios of Th2/Th1. The percentages of Th1, Th2 and Tc1 were comparable before and after immunotherapy within the rhinitic patient group. CONCLUSIONS: The preseasonal allergen subcutaneous immunotherapy applied to intermittent allergic rhinitis patients suppressed the percentage of IL-4 producing CD3+CD8+ cells. Decreased number of CD3+CD8+IL-4+ cells may participate in the regulatory mechanisms of immunotherapy.     

硫化镍与苯并芘转化人支气管上皮细胞基因差异表达谱的研究

目的用cDNA微阵列技术探讨经硫化镍(NiS)与二羟环氧苯并芘(dihydroxyepoxy benzo pyrene,BPDE)转化后的人支气管上皮细胞(16HBE)基因的差异表达;以了解不同种类和性质的化合物在转化细胞及致癌分子机制方面的异同;为预防人类生存环境不同外来化合物对人类健康的危害和影响提供科学依据.方法取16HBE分为NiS处理组,BPDE处理组和16HBE(对照组)三组同步培养,传代至第35代;提取三种细胞的总RNA,逆转录合成cDNA并以Cy3-dCTP和Cy5-dCTP荧光素分别标记制作探针.探针混合后与含4000个人类基因的芯片杂交.以ScanArray 4000扫描仪扫描芯片,以GenPix Pro3.0软件分析荧光信号,对三组差异表达的基因进行生物学信息分析.结果 NiS转化的16HBE细胞与BPDE转化的16HBE细胞经分析比较,呈现差异表达的基因有22个,其中11个(50%)下调,另11个(50%)上调.结论 NiS与BPDE对16HBE细胞株的转化作用在抑癌基因,细胞周期蛋白相关基因,细胞凋亡和应激反应基因相关基因,DNA合成、修复和重组蛋白相关基因,DNA结合、转录和转录因子类基因,细胞受体相关基因,代谢相关基因,蛋白翻译和合成相关基因等多类基因上呈现差异表达.认为基因芯片技术在筛选不同种类和性质的化合物转化细胞相关基因的改变上,具有高通量、高敏感、快速等特点,对化合物在细胞、分子毒作用和致癌机制的研究中意义重大.     

磺胺二甲嘧啶对FRTL-5细胞基因表达谱的影响

目的研究磺胺二甲嘧啶对FRTL5细胞基因表达谱的影响,探讨环境甲状腺素干扰物的作用机制。方法指数生长期细胞经20μgml磺胺二甲嘧啶处理24h后,用TRIzol法提取RNA,用9753位点的cDNA芯片检测基因表达情况,实验组和对照组细胞分别用Cy3dCTP和Cy5dCTP标记,计算Cy3和Cy5的比值,找出差异表达基因。结果芯片经扫描分析后,发现表达有差异的基因共有679条占芯片上基因总数的70%,其中395(40%)条基因表达增加,284(30%)条基因表达下降,涉及基因表达调控、细胞周期调控、细胞免疫,细胞代谢等众多事件。结论磺胺二甲嘧啶对FRTL-5细胞的毒性效应,其机制涉及多种基因。     

Detecting imperative genes and infiltrating immune cells in chronic Chagas cardiomyopathy by bioinformatics analysis

Chronic Chagas cardiomyopathy (CCC) is an acquired inflammatory cardiomyopathy triggered by the protozoan Trypanosoma cruzi infection. Although microvascular and neurogenic dysfunction and inflammation with persistent parasite presence in the heart may play a major pathogenetic role, little is known about the overall picture of gene co-expression regulating CCC. In this study, we aimed to explore the key biological pathways, hub genes and the landscope of infiltrating immune cells associated with inflammation in chronic Chagas cardiomyopathy. A weighted gene co-expression network analysis (WGCNA) was conducted based on the gene expression profiles from patients with and without chronic Chagas cardiomyopathy (GSE84796). Twelve coexpression modules were identified from the top 25% variant genes. Among them, the turquoise and black module were significantly positively correlated with CCC, which were highly enriched in Th1 and Th2 cell differentiation, the Cytokine-cytokine receptor interaction,NF-kappa B signaling pathway and T cell receptor signaling pathway. In addition, four genes (TBX21, ZAP70,IL2RB and CD69) were selected as candidate hub genes. Gene expression for hub genes were higher in CCC tissues compared to tissues from healthy controls. Additionally, gene set enrichment analysis (GSEA) analysis showed that high expressions of these hub genes were significantly correlated with interferon α response and interferon γ response. The microarray dataset GSE41089 further confirmed that although CD69 was not detected, the expression of TBX21, IL2RB and ZAP70 was also significantly up-regulated in the CCC mice compared to controls. We further studied the immune cells infiltration in CCC patients with CIBERSORT. The fraction of Mast cells activated,T cells CD8 and T cells gamma delta were significantly increased in CCC patients compared with control. Our research provides a more effective understanding of the pathogenesis of CCC and could help in future strategies for new diagnostic and therapeutic approaches for CCC patients.