细粒棘球蚴囊液对肝细胞表面TLR4表达的调节

目的 通过研究细粒棘球绦虫囊液对人肝细胞中Toll样受体(TLR)4表达的影响,探讨TLR在启动囊液抗细粒棘球绦虫信号转导通路中的作用.方法 用定量PCR检测肝细胞7404囊液处理前后TLR4和TGF-β1的表达变化.结果 肝细胞7404经囊液处理后随剂量增加TLR4表达减弱,TGF-β1随剂量增加表达增强,TLR2表达在各组均很低.结论 囊液可以上调TLR4的表达,提示囊液诱导的抗细粒棘球绦虫的信号转导途径町能由TLR4引起.高剂量囊液有助于TGF-β1所致的免疫逃避作用.     

PDGF、TNF-α在人肝细粒棘球蚴囊壁周围组织的分层表达

采用原位杂交方法检测血小板衍生生长因子 (PDGF )及肿瘤坏死因子α(TNF α )在 4 0例人肝细粒棘球蚴囊肿周围纤维囊壁中表达。结果显示PDGF、TNF α在肝细粒棘球蚴囊肿周围纤维囊壁中出现特异性分层表达。靠近虫体侧纤维囊壁中 ,PDGF、TNF α阳性细胞率分别为 (9 36 %± 2 13% )、 (10 5 2 %± 2 6 4 % )。靠近肝实质侧纤维囊壁中 ,PDGF、TNF α阳性细胞率分别为 (37 5 1%± 7 4 5 % )、 (2 5 76 %± 5 0 5 % )。PDGF、TNF α在两层中表达的均有显著性差异 (P <0 0 1)。同时 ,靠近肝实质侧纤维囊壁中PDGF与TNF α的表达也有显著性差异 (P <0 0 5 )。提示肝细粒棘球蚴囊肿周围人体纤维囊壁分层 ,PDGF、TNF α与肝实质侧纤维囊壁的形成有密切关系。     

乳腺细粒棘球蚴病1例

患者,女,44岁,因“左乳隐痛10余年,左乳肿块1月余”以“左乳肿物性质待查”入院。体检：左乳内上象限10-11点距乳晕5cm处可触及一约1．0cm×2．0cm大小的肿块,质硬,活动欠佳,边界尚清,有压痛。于人院后第3天行左乳肿块切除术。术中见左胸大肌筋膜近乳腺有一直径1．5cm大小的囊性包块,切除送检。[第一段]     

miRNA作为骨棘球蚴病诊断标志物的研究进展

骨棘球蚴病通常由细粒棘球绦虫和多房棘球绦虫引起,人类在食用被虫卵污染的食物或水时被感染,骨棘球蚴病的治疗一般包括手术和药物治疗,但治疗时间长、费用高,给患者造成了沉重负担。微小RNA(miRNA)已知参与多种生物过程和宿主-寄主相互作用,包括发育、细胞生长和死亡、寿命的相关靶点调节、转录、信号转导和细胞运动,这将有助于研究人员找到治疗和控制骨棘球蚴病的新策略和靶点。为进一步了解骨棘球蚴病,认清棘球绦虫在最终宿主和中间宿主中发育过程的分子基础至关重要,在细粒棘球绦虫、多房棘球绦虫中发现的miRNA在其各自宿主的表达调控中具有基因和发育阶段特异性。主要对miRNA作为骨棘球蚴病诊断标志物的研究进展进行综述。     

细粒棘球绦虫疫苗候选分子EgA31的GST融合蛋白原核表达及纯化

目的 :构建细粒棘球绦虫疫苗候选分子EgA31重组质粒 ,表达及纯化EgA31 GST融合蛋白 ,为疫苗的研究奠定基础。方法 :PCR扩增EgA31cDNA ,将得到的cDNA经限制性内切酶酶切 ,然后亚克隆入pGEX 5X 3质粒 ,转化BL2 1宿主菌 ,IPTG诱导重组质粒的表达 ,亲和层析纯化表达产物 ,Bradford法测定重组蛋白含量 ,用SDS PAGE和Westernblot进行分析鉴定。结果 :SDS PAGE显示分离纯化的EgA31 GST融合蛋白为 4 5kDa ,测序检验重组质粒中EgA31cDNA序列正确。EgA31 GST融合蛋白免疫豚鼠得到的抗血清可与细粒棘球绦虫原头蚴总蛋白在 6 6kDa处特异性反应。结论 :EgA31 GST融合蛋白获得高效表达 ,并成功纯化 ,初步实验证明具有良好的免疫原性 ,可用于进一步疫苗的免疫注射实验     

细粒棘球蚴感染小鼠早期阶段Tim-3表达的初步分析

目的:了解Tim-3作为一个新型促炎因子,在细粒棘球蚴感染小鼠的早期阶段的表达水平。方法:原头蚴感染BALB/c小鼠后1、5、9、13天,取其腹腔巨噬细胞和脾细胞。采用流式细胞术(FCM)检测各时间点腹腔巨噬细胞、脾巨噬细胞、脾CD3+细胞Tim-3的表达水平。采用qRT-PCR法检测各时间点TLR4的mRNA水平。结果:细粒棘球蚴组与对照组小鼠CD3+脾细胞Tim-3表达无明显差别;小鼠脾脏巨噬细胞(9、13天)、腹腔巨噬细胞(5、9、13天)表面Tim-3表达水平较对照组有升高,与对照组有显著差异(P<0.05)。巨噬细胞数量无明显变化。小鼠感染细粒棘球蚴第1天时TLR4 mRNA的表达水平升高,并与对照组有显著差异(P<0.05)。结论:小鼠感染细粒棘球蚴早期,巨噬细胞Tim-3的表达升高,并可能通过下调TLR4的表达,抑制了巨噬细胞功能,从而抑制宿主的免疫杀伤,产生了免疫逃避作用。     

细粒棘球绦虫重组BCG-Eg95疫苗的构建及其表达效率研究

目的 构建细粒棘球绦虫重组BCG-Eg95疫苗,分析Eg95分子在该疫苗中的表达效率.方法超声粉碎细粒棘球蚴组织提取总RNA,通过RT-PCR扩增Eg95的抗原编码基因;将该基因定向克隆到大肠杆菌-分枝杆菌穿梭表达载体pB-CG,构建重组质粒pBCG-Eg95;电穿孔法转化BCG,构建细粒棘球绦虫重组BCG-Eg95疫苗.免疫印迹分析重组BCG-Eg95疫苗的表达产物.结果 RT-PCR成功扩增出471 bp的Eg95抗原编码基因;双酶切证实Eg95抗原编码基因成功插入pBCG中;PCR证实rBCG-Eg95疫苗构建成功;免疫印迹分析发现重组BCG-Eg95疫苗的表达产物在相对分子质量(Mr)约为16.5×103处有明显的目的 蛋白表达条带,且能被感染细粒棘球蚴的鼠血清特异识别.结论 成功构建了细粒棘球绦虫重组BCG-Eg95疫苗,为而后的开发利用奠定了理论基础.     

TLR2及TLR4在侵袭性肺曲霉病小鼠中的表达

目的 研究Toll样受体(TLR)2和TLR4在侵袭性肺曲霉病小鼠中的表达,探讨TLR2和TLR4在侵袭性肺曲霉病中的作用. 方法 将小鼠分为3组:A组为正常对照组;B组为未免疫抑制但感染烟曲霉菌组;c组为侵袭性肺曲霉病(IPA)模型组,给予免疫抑制并感染烟曲霉菌.在感染后8、24、48和72 h时相点,处死小鼠,取肺组织,采用组织病理学方法观察肺组织的病理损伤,RT-PCR法检测肺组织各个时相点的TLR2、TLR4、TNF.ot和β-tublin的表达.TLR2、TLR4、TNF-α的PCR产物电泳条带的扫描密度值与同时扩增的β-tublin电泳条带的扫描密度值的比值用以表示TLR2、TLR4和TNF-α的相对表达水平. 结果 病理观察结果显示,对照组小鼠的肺组织结构正常;正常小鼠感染烟曲霉菌后,小鼠的肺组织有炎症细胞浸润、出血等炎症反应,但未见孢子萌芽生成菌丝;而IPA模型小鼠的肺组织病理损伤严重,可见炎症细胞浸润、肺泡塌陷伴随出血,孢子聚积并萌芽生成菌丝.8、24、48 h 3个时间点的TLR4和24、48 h两个时相点的TNF-α在IPA模型小鼠肺组织中的表达要低于烟曲霉菌感染的正常小鼠(P<0.05),而TLR2在烟曲霉菌感染的正常小鼠和IPA模型小鼠肺组织中呈现低表达,但24、72 h时相点的TLR2在IPA模型小鼠的表达要低于烟曲霉菌感染的正常小鼠(P<0.05). 结论 TLR4及其下游分子TNF-α在IPA模型小鼠肺组织中低表达,在组织病理镜检中可见肺曲霉病典型肺组织病理损伤和孢子生成菌丝.     

细粒棘球绦虫重组BCG-Eg95疫苗诱导的保护力观察

目的 探讨细粒棘球绦虫重组BCG-Eg95疫苗免疫鼠后对Eg原头节攻击感染的保护性作用.方法 '将细粒棘球绦虫重组BCG-Eg95疫苗采用皮下注射、鼻腔内接种、口服灌胃和肌肉注射4种途径分别免疫Balb/C鼠,免疫后8W用Eg 原头节进行攻击感染,感染后18周剖杀小鼠,计算减蚴率,测定血清中IgG及其亚类和IgE水平,同时设有BCG和PBS对照.结果 疫苗接种组的减蚴率为18.20%～92.46%,血清IgG、IgG2a、IgG2b水平明显升高,IgG1、IgG3和IgE显著降低.结论 细粒棘球绦虫重组BCG-Eg95疫苗口服灌胃和肌肉注射是两种较好的接种途径,IgG、IgG2a和IgG2b在疫苗诱导的保护力中起重要作用.     

细粒棘球蚴AgB亚单位基因的联合表达和血清学评价

目的为进一步提高AgB抗原在诊断中的敏感性和特异性,将细粒棘球蚴AgB1和AgB2两个亚单位基因在同一载体中进行联合表达,并对联合表达的重组抗原和单基因表达的AgB1、AgB2在血清抗体检测中的差异进行比较研究。方法在表达载体PET32a中构建AgB1＋AgB2（AgBs）联合基因的重组质粒,用ELISA检测重组抗原与病人血清的反应性。结果构建了AgBs重组质粒,经测序证实插入的联合基因片段序列正确。AgBs重组质粒表达的融合蛋白分子量为38kDa,为不溶性蛋白。联合表达抗原AgBs对细粒棘球蚴病（CE）血清的敏感性为84．4％,特异性为80．5％;单基因表达的AgB1和AgB2的敏感性分别为75．6％和57．8％,特异性分别为72．6％和91．2％。结论成功构建了AgB亚单位联合表达基因,联合表达的AgBs抗原对CE血清的诊断价值优于单基因表达的AgB1或AgB2抗原。     

Cytotoxicity ofEchinococcus granulosus cyst fluid in vitro

In a cytotoxicity assay, using rat spleen cells as target cells,Echinococcus granulosus cyst fluid from cattle exerted a marked degree of cytotoxicity in vitro. When trypan blue exclusion or [³H]thymidine incorporation by concanavalin A stimulated spleen cells was used as a measure of cell viability, dialyzed cyst fluid showed maximum cell destruction up to a 1:8 dilution. The effect was dose and time dependent, cells being affected by 24 h after exposure to cyst fluid. The components responsible for cytotoxicity of cyst fluid were heat stable and could be recovered using gel chromatography on Sephadex G 50 and G 15 as a single low molecular weight fraction. It is assumed that the toxic products released by the living parasite can readily penetrate through the cyst wall into the surrounding host tissue. The interference of such substances with immunocompetent cells might account for the long-term survival of the parasite in the intermediate host.Dedicated to Prof. Dr. G. Piekarski on the occasion of his 70th birthday     

Modulation of dendritic cell differentiation and cytokine secretion by the hydatid cyst fluid of Echinococcus granulosus

Chronic infection by Echinococcus granulosus results in establishment of fluid-filled cysts (hydatid cysts) in liver or lungs of infected hosts, which can escape destruction by the host immune system for long periods. This study explores the modulation by hydatid cyst fluid of the in vitro human monocyte to dendritic cell (DC) transition induced by granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). Addition of the fluid to adherent peripheral blood monocytes cultured in GM-CSF/IL-4 stimulates release of prostaglandin E2 (PGE2) and IL-6. Exposure of differentiating DC to the fluid during the 7-day culture in GM-CSF/IL-4 impairs their subsequent ability to secrete IL-12, IL-6 or PGE2 in response to lipopolysaccharide (LPS) stimulation. This inhibition is not dependent on the initial release of PGE2. The presence of hydatid cyst fluid also modulates the phenotype of the cells generated during culture, resulting in increased CD14 expression and decreased expression of CD1a. Finally, hydatid fluid can stimulate predifferentiated DC to mature, as evidenced by release of IL-12 and IL-6, and by up-regulation of class II major histocompatibility complex and CD86. The possible role of dendritic cell modulation in regulating the host immune response to hydatid cysts is discussed.     

TLR2 and TLR4 expression on the immune cells of tuberculous pleural fluid

Toll-like receptors (TLRs) play an important role in mediating the down stream signaling of immune response in tuberculosis. The predominance of Th1 response in tuberculous pleurisy prompted us to study the expression profiles of TLR2 and TLR4 on different immune cells and on subsets of T cells obtained from the site of infection. Our results showed that TLR2 was up-regulated on the monocytes from pleural fluid indicating a prominent role for this receptor in anti-tuberculous immunity. Notably, TLR2 and TLR4 expression were also enhanced on IFN-gamma secreting CD4(+)T cells. However, their expression was down-regulated on activated and IL-4 secreting CD4(+)T cells from the site of infection indicating that TLR expression is differentially modulated on the different subsets of T cells depending on their activation status and cytokine expression. The down-regulation of both TLRs on the natural regulatory T cells despite their higher number at the site of infection might be a mechanism to maintain their suppressive activity.     

Specific and cross-reactive antigens of Echinococcus granulosus hydatid cyst fluid

The parasite antigens of Echinococcus granulosus hydatid cyst fluid have been characterised using the techniques of radioiodination, immunoprecipitation, SDS-PAGE, and immunoblotting. Five major subunit antigens of the parasite have been identified of relative molecular mass (Mr) 12,000, 16,000, two at 20,000, and 38,000 when reduced. The 12 and 16 kDa molecules are specific to E. granulosus and are excreted/secreted by both U.K. strains of the parasite and all human isolates examined, although not all hydatid disease patients produce antibodies to them. These molecules may be suitable for detection in specific immunodiagnosis. The 38 kDa molecule associates, via a disulphide linkage, with one of the 20 kDa molecules to form a single molecule of 60 kDa. This antigen is cross-reactive with human antibody to other cestode, trematode, and nematode parasites. Part of this cross-reactivity is associated with the presence of phosphorylcholine bound to the 38 kDa subunit.     

Toll样受体2在人胎盘和胎膜组织中的表达

目的 检测正常胎盘和胎膜组织中Toll样受体2(TLR2)的表达.方法 收集5例足月剖宫分娩胎盘和胎膜样本,运用RT-PCR法检测胎盘和胎膜组织中TLR2 mRNA的表达,运用免疫组织化学和Westen blot印迹方法检测TLR2蛋白质在胎盘和胎膜组织中的表达.结果 RT-PCR显示在mRNA水平,胎盘和胎膜组织中均有TLR2表达;Westen blot印迹显示TLR2抗体在胎盘和胎膜组织中相对分子质量为50000左右的位置有一条明显的条带;免疫组化研究显示TLR2在胎盘的合体滋养细胞、胎膜的羊膜上皮细胞及平滑绒毛膜滋养细胞中有表达.结论 TLR2在人胎盘和胎膜组织中的表达,提示其在妊娠期先天性免疫中可能发挥重要作用.     

Toll样受体2在人胎盘和胎膜组织中的表达

目的 检测正常胎盘和胎膜组织中Toll样受体2(TLR2)的表达.方法 收集5例足月剖宫分娩胎盘和胎膜样本,运用RT-PCR法检测胎盘和胎膜组织中TLR2 mRNA的表达,运用免疫组织化学和Westen blot印迹方法检测TLR2蛋白质在胎盘和胎膜组织中的表达.结果 RT-PCR显示在mRNA水平,胎盘和胎膜组织中均有TLR2表达;Westen blot印迹显示TLR2抗体在胎盘和胎膜组织中相对分子质量为50000左右的位置有一条明显的条带;免疫组化研究显示TLR2在胎盘的合体滋养细胞、胎膜的羊膜上皮细胞及平滑绒毛膜滋养细胞中有表达.结论 TLR2在人胎盘和胎膜组织中的表达,提示其在妊娠期先天性免疫中可能发挥重要作用.     

Echinococcus granulosus: occurrence of ecdysteroids in protoscoleces and hydatid cyst fluid

Both free ecdysteroids and hydrolysable polar conjugated ecdysteroids were detected in protoscoleces of Echinococcus granulosus from the equine host, and in hydatid cyst fluid from the same source. Comparisons were made of hydatid cyst fluid from E. granulosus infections of three intermediate host species: horses, sheep and humans. Ecdysone and 20-hydroxyecdysone were identified in both protoscoleces and hydatid cyst fluids by high-performance liquid chromatography monitoring fractions by radioimmunoassay, and by capillary gas chromatography/mass spectrometry (selected ion monitoring). The free ecdysteroid fractions of hydatid cyst fluid from horses and sheep also contained several unidentified, chromatographically unique, immunoreactive compounds which were refractory to hydrolysis with a crude Helix pomatia aryl sulphatase enzyme preparation.     

Toll样受体2在人胎盘和胎膜组织中的表达

目的 检测正常胎盘和胎膜组织中Toll样受体2(TLR2)的表达.方法 收集5例足月剖宫分娩胎盘和胎膜样本,运用RT-PCR法检测胎盘和胎膜组织中TLR2 mRNA的表达,运用免疫组织化学和Westen blot印迹方法检测TLR2蛋白质在胎盘和胎膜组织中的表达.结果 RT-PCR显示在mRNA水平,胎盘和胎膜组织中均有TLR2表达;Westen blot印迹显示TLR2抗体在胎盘和胎膜组织中相对分子质量为50000左右的位置有一条明显的条带;免疫组化研究显示TLR2在胎盘的合体滋养细胞、胎膜的羊膜上皮细胞及平滑绒毛膜滋养细胞中有表达.结论 TLR2在人胎盘和胎膜组织中的表达,提示其在妊娠期先天性免疫中可能发挥重要作用.