Staphylococcus aureus enterotoxin B-induced endoplasmic reticulum stress response is associated with chronic rhinosinusitis with nasal polyposis

ObjectiveStaphylococcus aureus enterotoxin B (SEB) might participate in the pathogenesis of chronic rhinosinusitis with nasal polyps (CRSwNP). However, the exact mechanism of polyp formation in CRSwNP remains unclear. Since the endoplasmic reticulum (ER) stress response is closely associated with chronic inflammation, we investigated the association between ER stress and SEB in the pathogenesis of CRSwNP.Design and methodsTwenty-three CRSwNP patients with eosinophilic polyps (EP) or non-eosinophilic polyps (NEP) and 10 healthy subjects who were undergoing septoplasty were enrolled in this study. ER stress response was investigated using immunohistochemical staining and Western blotting.ResultsWe show in this study that there are significantly more SEB-positive cells and higher production of reactive oxygen species (ROS) in the epithelial layer of EP than NEP or control tissue. Both SEB and protein A were detected strongly in tissues from patients with CRSwNP. We observed SEB induced the ER stress response in RPMI 2650 cells. GRP78 elevation by SEB was reduced by ROS scavenger pretreatment. In addition, the induction of GRP78 and p47 phox was increased significantly in EP compared with NEP or control mucosa.ConclusionsSEB may induce ER stress via ROS production in CRSwNP. Therefore, we suggest that SEB-induced ER stress may play important roles in the pathogenesis of nasal polyposis.     

金黄色葡萄球菌肠毒素A基因克隆及表达

目的对金黄色葡萄球菌肠毒素A基因(SEA)进行克隆、鉴定与表达,为制备单抗、诊断试剂及进一步深入研究SEA的功能奠定基础。方法用聚合酶链反应(PCR)扩增SEA基因,将扩增的产物连接于测序载体pMD18-T上,经测序反应确定无误,酶切后将SEA基因与原核表达载体pET42b( )构建表达SEA的重组质粒,将重组质粒先转化入大肠杆菌DH5α内并提取质粒,经双酶切鉴定后,再转化入表达宿主大肠杆菌BL21菌株内,对转化菌株进行诱导后,破菌,进行十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)。结果构建了表达载体pET42b( )-SEA,并获得高效表达,表达的蛋白质相对分子质量为27 000,重组蛋白(rSEA)在37℃、25℃经异丙基硫化-β-D半乳糖苷(IPTG)诱导时均以包涵体形式存在。结论SEA基因成功克隆到表达质粒内并表达,为制备单抗、诊断试剂及其致病机制研究奠定了基础。     

金黄色葡萄球菌肠毒素A基因克隆及表达

目的 对金黄色葡萄球菌肠毒素A基因（SEA）进行克隆、鉴定与表达,为制备单抗、诊断试剂及进一步深入研究SEA的功能奠定基础。方法 用聚合酶链反应（PCR）扩增SEA基因,将扩增的产物连接于测序载体pMD18-T上,经测序反应确定无误,酶切后将SEA基因与原核表达载体pET42b（＋）构建表达SEA的重组质粒。将重组质粒先转化入大肠杆菌DH5a内并提取质粒,经双酶切鉴定后,再转化入表达宿主大肠杆菌B121菌株内,对转化菌株进行诱导后,破菌,进行十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）。结果 构建了表达载体pET42b（＋）-SEA,并获得高效表达,表达的蛋白质相对分子质量为27000,重组蛋白（rSEA）在37℃、25％经异丙基硫化-β-D半乳糖苷（IPTG）诱导时均以包涵体形式存在。结论 SEA基因成功克隆到表达质粒内并表达,为制备单抗、诊断试剂及其致病机制研究奠定了基础。     

鼻后神经切断术治疗慢性鼻窦炎伴鼻息肉合并哮喘的疗效及其对鼻黏膜嗜酸性粒细胞的影响

目的 探讨鼻后神经切断术治疗慢性鼻窦炎伴鼻息肉(CRSwNP)合并哮喘的疗效,以及手术对鼻黏膜内嗜酸性粒细胞(EOS)的影响.方法 选取该院确诊的CRSwNP合并哮喘患者15例,行鼻后神经切断术.术后随访1年,采用视觉模拟法(VAS)评估疗效.有7例患者术前和术后1年采集了中鼻甲黏膜,比较手术前后鼻腔黏膜EOS浸润度的...     

鼻窦炎鼻内镜手术疗效及影响因素

目的 探讨影响鼻窦炎鼻内镜手术疗效的相关因素,旨在提高其手术远期疗效.方法 对226例(352侧)慢性鼻窦炎及鼻息内全身麻醉下功能性鼻内镜鼻腔鼻窦手术病例的手术效果及影响因素进行了分析,随访1年以上.结果 Ⅰ、Ⅱ、Ⅲ型鼻窦炎的有效率分别为97.5%、87.9%和74.4%,各型鼻窦炎有效率差异均具有统计学意义(P＜0.05或P＜O.01),术后能坚持定期随诊和规范化综合治疗组与未能坚持随诊和规范化综合治疗组的有效率分别为97.2%和43.1%,其差异有统计学意义(P＜0.01).结论 对鼻窦炎及鼻息肉应在其早期采取积极的手术治疗,术后坚持定期随诊和规范化综合治疗是获得较好的远期疗效的重要保证.     

鼻内镜术后患者认知功能障碍的影响因素分析

目的 探讨鼻内镜术后患者认知功能障碍的影响因素。 方法 回顾分析2016年8月-2018年8月在耳鼻咽喉科全身麻醉下行鼻内镜手术的113例患者病历资料,按照自行设计的鼻内镜术后认知功能障碍(postoperative cognitive dlhgys-function , POCD)危险因素调查表收集患者相关信息,分析不同特征人群的术后POCD的发生情况,并通过Logistic回归分析鼻内镜术后POCD发生的独立影响因素。 结果 113例顺利完成鼻内镜术的患者中,28例患者术后出现POCD;Logistic回归分析显示年龄、动脉硬化危险因素个数、文化程度、高血压病是鼻内镜术后POCD发生的独立危险因素。 结论 影响鼻内镜术后POCD发生的危险因素较多,需特别注意动脉硬化危险因素数多及老年患者。     

表皮葡萄球菌在慢性鼻-鼻窦炎患者中生物膜的表达

目的研究表皮葡萄球菌生物膜在慢性鼻-鼻窦炎(CRS)患者鼻黏膜中的表达情况。方法将临床手术中获取的180例慢性鼻-鼻窦炎患者内镜手术黏膜样本,根据细菌培养结果将其分为4组,分别为表皮葡萄球菌生长组79例,其他葡萄球菌生长组33例,阴性杆菌生长组27例,细菌培养阴性组41例,所有样本均行标准的扫描电子显微镜方法检测。结果表皮葡萄球菌生长组79例全部观察到生物膜的存在,表达率为100%(79/79),其他葡萄球菌生长组为75.8%(25/33),阴性杆菌生长组为77.8%(21/27),细菌培养阴性组为41.5%(17/41)。后3组分别与表皮葡萄球菌组比较,P值均<0.01。结论表皮葡萄球菌在CRS患者生物膜形成中起重要作用。     

表皮葡萄球菌在慢性鼻-鼻窦炎患者中生物膜的表达

目的研究表皮葡萄球菌生物膜在慢性鼻-鼻窦炎（CRS）患者鼻黏膜中的表达情况。方法将临床手术中获取的180例慢性鼻-鼻窦炎患者内镜手术黏膜样本,根据细菌培养结果将其分为4组,分别为表皮葡萄球菌生长组79例,其他葡萄球菌生长组33例,阴性杆菌生长组27例,细菌培养阴性组41例,所有样本均行标准的扫描电子显微镜方法检测。结果表皮葡萄球菌生长组79例全部观察到生物膜的存在,表达率为100%（79/79）,其他葡萄球菌生长组为75.8%（25/33）,阴性杆菌生长组为77.8%（21/27）,细菌培养阴性组为41.5%（17/41）。后3组分别与表皮葡萄球菌组比较,P值均〈0.01。结论表皮葡萄球菌在CRS患者生物膜形成中起重要作用。     

鼻内激素联合鼻腔冲洗在儿童慢性鼻-鼻窦炎治疗中的疗效观察

目的：观察鼻内激素联合鼻腔冲洗在儿童慢性鼻-鼻窦炎治疗的疗效。方法选取2009年10月至2012年10月收治的慢性鼻-鼻窦炎80例,随机分为两组,每组40例。试验组患儿采用鼻内激素联合鼻腔冲洗治疗,对照组患儿采用单纯的常规鼻内激素治疗,不行鼻腔冲洗。比较两组患儿用药1个月后的临床疗效和症状缓解时间。结果试验组患儿治疗后总有效率（95.0%）明显高于对照组（60%）,且其鼻塞、流鼻涕、头晕、头痛等症状缓解时间也明显优于对照组,差异有统计学意义（ P<0.05）。结论鼻内激素联合鼻腔冲洗相较于常规鼻内激素治疗儿童慢性鼻-鼻窦炎效果明显,其临床症状缓解时间良好,不良反应少,有一定的临床优势,值得推广使用。     

超抗原SEB或SEC对T细胞增殖和活化的影响

目的:探讨超抗原金黄色葡萄球菌肠毒素B(staphy lococcal enterotoxinB,SEB)和金黄色葡萄球菌肠毒素C(staphy lococcal enterotoxinC,SEC)在体外对T细胞增殖和活化的影响。方法:不同浓度SEB或SEC在体外刺激T细胞,采用MTT法检测T细胞增殖水平,在流式细胞仪(flowcytometer,FCM)上检测T细胞受不同浓度SEB或SEC刺激后不同时间其早期活化标志CD69的表达水平。结果:SEB或SEC在体外能明显激活T细胞,以浓度为100μg/L时活化T细胞的作用最强,刺激时间以4～8h为佳,而对T细胞的增殖无明显影响。SEB和SEC活化T细胞的作用强度之间无明显差异。结论:以上结果提示,SEB或SEC具有很强的刺激T细胞活化的能力,为其应用于肿瘤免疫治疗提供了一定依据。     

金黄色葡萄球菌肠毒素B基因在大肠杆菌中的克隆及表达

目的扩增金黄色葡萄球菌肠毒素B(SEB)基因,构建其原核表达载体,并进行诱导、表达,为其应用研究奠定基础。方法根据GenBank中SEB的基因序列,设计一对分别含BamHⅠ、XhoⅠ酶切位点的特异性引物,以金黄色葡萄球菌基因组DNA为模板进行PCR扩增后,经BamHⅠ、XhoⅠ双酶切,并与做相应酶切的pET-28α(+)连接,转化大肠杆菌BL21,提取质粒进行双酶切鉴定及测序,用IPTG诱导表达融合蛋白,SDS-PAGE和Western blot印迹鉴定表达产物。结果成功扩增出SEB基因,基因大小为801bp,重组PET-28α(+)-SEB双酶切鉴定可见目的片段,测序结果显示SEB在正确读框中,序列比对分析显示其与相关报道核苷酸序列一致性达99%。经IPTG诱导后,pET-28α(+)-SEB/BL21在相应的相对分子质量(35×103)可见融合蛋白以包涵体形式表达,免疫印迹在相应分子量检测到目的蛋白。结论克隆了SEB基因,并成功在大肠杆菌BL21中以包涵体形式表达,为肠毒素B应用研究奠定了基础。     

A simple and rapid fluorescence-based immunoassay for the detection of staphylococcal enterotoxin B

The bioterrorism threat is perceived to be a real challenge to our nation's security. This threat has necessitated the design of better and faster assays for the detection of biothreat agents including staphylococcal enterotoxin B (SEB), a causative agent of food poisoning. This study describes a simple, fast and highly sensitive fluorescence-based immunoassay, in which the antibody is fluorescently-labeled for use in this assay. Use of labeled antibodies resulted in very low level of detection of SEB, 100 pg/well. This method is four times faster than classical and conventional enzyme-linked immunosorbent assay (ELISA).     

DIP内镜评分方案在评价行鼻内镜手术的慢性鼻-鼻窦炎患者病情方面的应用

目的探讨DIP内镜评分方案在慢性鼻-鼻窦炎(CRS)行鼻内镜手术中的价值。方法选取2013年9月-2015年2月该院收治行鼻内镜手术的82例CRS患者为研究对象,术前进行Lund-Mackay CT评分、鼻腔鼻窦结局测量20条(SNOT-20)及视觉模拟量表(VAS)评分,术中录制鼻内镜检查录像。术后3个月再次进行SNOT-20和VAS评分,再次录制鼻内镜录像。由两名医生作为评分者对所有录像进行DIP及Lund-Kennedy内镜评分,采用组内相关系数(ICC)来比较两者的信度,分析各评分结果之间的相关性。结果术后SNOT-20、VAS评分、DIP及Lund-Kennedy内镜评分均明显低于术前(P0.05)。术前DIP内镜评分与Lund-Kennedy内镜评分、Lund-Mackay CT评分均成正相关(P0.05),与SNOT-20及VAS评分均无明显的相关性(P0.05)。术后DIP内镜评分与Lund-Kennedy内镜评分成正相关(P0.05),与SNOT-20及VAS评分均无明显的相关性(P0.05)。手术前后DIP内镜评分的评分者信度及重测信度的ICC均高于Lund-Kennedy内镜评分。结论 DIP内镜评分在CRS患者鼻内镜手术前后均有较高的信度,与其他客观评估结果呈正相关,而与主观评分结果无明显相关,故需从主观和客观两个方面来综合评估CRS的病情及疗效。     

A cytotoxic substance (CTS-51) produced by human buffy coat cultures stimulated by staphylococcal enterotoxin B: further characterizations and combined action with interferon

A recently recognized unique cytotoxic substance, CTS-51, was tested for the hear or acid stability, trypsin digestion and dialysis. Moreover, influences of elevated incubation temperatures or serum concentrations of medium on the cytotoxic activity of CTS-51, and the combination effects of CTS-51 and human leucocyte interferon (HuIFN-alpha (Le)) were investigated. The cytotoxic activity of CTS-51, which is promoted by a small molecule easily passable the dialysis membrane, was found to be very stable to heat (even at 100 degrees C for 30 min) or acid (pH 2.0 for 24 hr at 4 degrees C) treatments. The treatment with 0.75% trypsin for 1 hr did not diminish the CTS-51 activity. The susceptibility of Daudi lymphoma cells to the antiproliferative action of HuIFN-alpha (Le) was further potentiated by treating the cells with CTS-51 for 16 hr. On the other hand, the CTS-51 activity which was revealed to be prescribed by its concentration in the medium, was not potentiated at 39 degrees C when compared to that at 37 degrees C in contrast to HuIFN-alpha (Le) action, and was reduced according to the increase of the fetal calf serum concentration in the medium.     

鼻后神经下鼻甲支切断术联合玉屏风颗粒治疗慢性鼻-鼻窦炎伴有鼻息肉合并哮喘的疗效分析*

目的 探讨鼻后神经下鼻甲支切断术联合玉屏风颗粒治疗慢性鼻-鼻窦炎伴有鼻息肉合并哮喘的疗效。方法 选取该院确诊的慢性鼻-鼻窦炎伴有鼻息肉合并哮喘的患者46例。分成两组,对照组(n=25)予以鼻窦开放和鼻息肉切除术,实验组(n=21)在常规手术基础上行鼻后神经下鼻甲支切断术。两组术后口服玉屏风颗粒治疗2个月。随访6个月,采用视觉模拟量表(VAS)和Lund-Mackay评分法进行疗效评估。结果 实验组术后6个月疗效明显高于对照组,两组比较,差异有统计学意义(χ~2=6.40,P=0.041)。结论 鼻后神经下鼻甲支切断术联合玉屏风颗粒治疗慢性鼻-鼻窦炎伴有鼻息肉合并哮喘,可有效提高临床疗效,提升患者生活质量。     

Inflammatory mediators and eosinophilia in atopic and non-atopic patients with nasal polyposis.

Nasal polyps are characterized by eosinophilic infiltration and presence of inflammatory mediators, such as total IgE, eosinophil cationic protein (ECP) and cytokines. The role of atopy in nasal polyp pathogenesis is still unclear. Therefore, we evaluated serum IgE levels, nasal mucus concentrations of ECP and cytokines and the number of infiltrating eosinophils in nasal tissue of polyps from atopic and non-atopic patients. Samples were obtained from a randomized population of 31 patients with nasal polyposis having endonasal sinus surgery and of 13 control subjects undergone corrective surgery of the nasal septum. On the basis of medical history of allergy, positive skin-prick tests and total IgE levels, patients with polyposis were divided in atopic (n = 13) and non-atopic (n = 18) patients. We determined levels of IgE in blood, ECP and cytokines (IL-4, IL-6, IL-8, IFN-gamma and IL-2) in nasal mucus, and number of infiltrating eosinophils in nasal tissue. The concentrations of total IgE, ECP, IL-4 and IL-8 and eosinophilia were significantly higher in all patients with nasal polyps compared with controls. Inside, all patients with nasal polyposis showed lower levels of IL-6, IFN-gamma and IL-2 compared with controls. The atopic patients showed significant differences when compared with non-atopic patients for the higher concentrations of total IgE (698.80+/-322.24 vs. 279.63+/-234.11; P < 0.0001) and IL-8 (1437.2 pg/ml+/-1250.7 vs. 605.5 pg/ml+/-481.1; P < 0.015). These findings suggest that inflammation still remains the major factor in the etiology of nasal polyposis and show different levels of inflammatory mediators into atopic and non-atopic patients.     

辅助性T细胞因子在慢性鼻-鼻窦炎和鼻息肉中的表达及意义

目的：探讨辅助性T细胞（T h细胞）因子白细胞介素（IL-4）、γ-干扰素（IFN-γ）在慢性鼻-鼻窦炎（CRS）、鼻息肉（NP）以及正常下鼻甲黏膜组织中表达的异同及其可能的作用。方法：采用酶联免疫吸附法（EL ISA法）检测IL-4和IFN-γ在CRS组（20例）、鼻息肉组（20例）和正常下鼻甲黏膜中（对照组15例）的表达,比较它们在不同组织中表达的差异。结果：IL-4只在NP组表达高于对照组（P〈0.001）;IFN-γ在NP组及CRS组表达均高于对照组（P〈0.05）,且以CRS组更为显著（P〈0.05）。结论：CRS,NP组织中显示出不同的IL-4,IFN-γ表达优势。