细菌膜囊泡的工程改造与应用

细菌膜囊泡 (Bacterial Membrane Vesicles,BMVs) 是原核细菌在生长繁殖过程中产生的一种囊泡状结构,直径在20~400 nm之间。BMVs的主要成分为细菌蛋白质、核酸以及脂类。由于BMVs中蛋白和/或膜脂的免疫原性,可有效刺激机体的固有免疫和适应性免疫系统,产生针对相应抗原的免疫能力;同时因 BMVs不含完整的基因组,无法自我增殖, 具有较高的生物安全性,因而在疫苗制备、抗原/药物递送系统、抗肿瘤制剂研发方面具有广阔的应用前景。野生型细菌产生的 BMVs通常产量较低,且携带内毒素等毒性成分,故常常需要对 BMVs产生菌或 BMVs本身进行工程改造,实现降低 BMVs毒性,提高BMVs产量,增强BMVs的免疫原性,促进工程化BMVs的开发应用。     

聚合物囊泡作为药物载体的研究进展

聚合物囊泡作为一类新型的药物载体,具有独特优越的结构、性质和稳定性,可包载多种亲水、疏水性药物分子,已经成为当前分子自组装纳米药物载体的研究热点.论述了聚合物囊泡的结构和特点、制备方法与形成条件,介绍了形成聚合物囊泡的两亲聚合物类型及所形成的聚合物囊泡的特性,并对聚合物囊泡作为药物载体在诊断和治疗中的研究进展作一综述.     

革兰阴性菌外膜囊泡在感染和免疫调节中的研究进展

革兰阴性菌释放的细胞外囊泡源于细胞最外层膜, 被称为外膜囊泡(outer membrane vesicle, OMV), 含有磷脂、脂多糖、外膜蛋白和细菌特异性抗原等细菌外膜成分。OMV在细菌生理学和致病机制中发挥重要作用, 可参与生物膜形成、基因水平转移、应激和炎症反应、毒素和其他生物分子的传递等, 且在免疫调节以及肠道微生物群的建立和平衡中也起着重要作用。本文对OMV在细菌感染及免疫调节中的作用进行综述, 为细菌导致的感染性疾病预防和治疗提供新的思路, 并对OMV在肿瘤靶向治疗和新型疫苗制备中的潜在应用价值进行展望。     

革兰氏阳性菌细胞外囊泡研究进展

细胞外囊泡（EVs）是一种包括真核细胞和细菌在内的多种细胞分泌的具有脂质双层结构的囊泡。革兰氏阴性菌分泌的EVs已被广泛研究多年,但革兰氏阳性菌由于细胞壁的结构特征,一直被认为无法分泌EVs。然而近年来的研究发现,革兰氏阳性菌同样可以分泌EVs,其分泌的EVs携带了蛋白质、核酸、脂质以及代谢物等多种组分,在革兰氏阳性菌的生物膜形成、耐药性、水平基因转移（HGT）及对宿主细胞的免疫调节等方面发挥重要作用,在疫苗研发、疾病治疗和药物递送等领域具有重要的潜在应用价值。本文通过对近年来革兰氏阳性菌EVs的相关研究进行综述,以期为革兰氏阳性菌EVs的相关研究提供参考。     

鲍曼不动杆菌外膜囊泡诱发人单-核巨噬细胞THP-1的炎性反应

目的 建立超速离心法纯化鲍曼不动杆菌外膜囊泡(OMVs),并探讨OMVs诱发人单核-巨噬细胞THP-1炎性反应能力。方法 大量培养鲍曼不动杆菌标准菌株,从培养液上清中以超速离心法提纯OMVs并以透射电镜观察形态。分别以5和50μg/mL的OMVs与THP-1细胞共培养,RT-qPCR检测NOD样受体家族蛋白(NLRP3)和caspase-1表达量;ELISA检测培养液上清中IL-1β含量;蛋白质印迹法检测细胞自噬相关蛋白LC-3Ⅱ和Beclin-1表达量;酶促动力学法检测细胞caspase-3酶活性;流式细胞测量术检测细胞活性氧表达量。结果 成功提纯鲍曼不动杆菌OMVs。OMVs可以诱导THP-1细胞NLRP3炎性复合体活化,并增加自噬相关蛋白LC-3Ⅱ和Beclin-1表达量(P<0.05),以及上调凋亡关键因子caspase-3酶活性(P<0.05)。共培养1 h时,OMVs可以显著上调THP-1细胞活性氧表达(P<0.05)。结论 鲍曼不动杆菌OMVs可以显著活化巨噬细胞NLRP3炎性复合体。     

聚合物囊泡在生物医学领域中的应用

聚合物囊泡是近年来新兴的一种自组装软性纳米材料.由于其优越的理化性质,聚合物囊泡已经受到了巨大的关注并且已经被利用在多个领域.文章综述了聚合物囊泡作为药物投递载体进行小分子化合物给药治疗肿瘤和反义核酸投递进行基因治疗;模拟细胞功能重建了ATP合成过程及模拟体内三酶偶联反应过程;作为近红外荧光探针在活体深层组织荧光成像技术等生物医学领域中的应用.     

胞外囊泡在恶性肿瘤发生发展中的作用

通过胞外囊泡(extracellular vesicles,Evs)进行的细胞内信号转导是一种被低估的细胞间的沟通方式.EVs携带很多生物活性分子、表面受体以及基因信息,例如蛋白编码基因和miRNAs,lncRNAs以及mRNAs都可通过EVs在细胞间传递并且影响受体细胞的功能.本篇文章阐述了EVs,尤其是EVs相关的miRNAs如何影响恶性肿瘤的发生、侵袭、转移和复发.     

胰原基伴囊泡肾、囊泡肾上腺等多项变异一例

胰原基伴囊泡肾、囊泡肾上腺等多项变异一例肖洪文泸州医学院泸州６４６０００在解剖一例约３０岁男尸时发现：（１）肝大，长约２５ｃｍ，左叶达左腋中线；（２）胰原基：腹背胰分离。腹胰呈一底向上的三角形，位于十二指肠降部内侧缘和肠系膜上血管之间，细小的腹胰管开...     

淋球菌外膜蛋白疫苗的研究进展

在自然界 ,淋球菌的唯一宿主是人类 ,淋球菌在漫长的进化过程中已经形成了一套逃避人体免疫系统的机制 ,加上缺少很好的动物实验模型 ,给制备淋球菌疫苗进行淋病预防造成相当大的障碍。但近年来 ,从Por,Pil,Opa和LOS等淋球菌外膜蛋白抗原的一系列研究中 ,人们认识到理想的淋球菌疫苗抗原应该具有的一些特性 ,有力地推动了淋球菌疫苗的研究。     

基于胞外囊泡治疗诊断学智能药物递送工具

胞外囊泡 （EVs） 作为细胞外的膜颗粒,在细胞间信号传导中起着至关重要的作用。研究表明,EVs有可能用作特定器官 （如内耳） 的生物标志物或药物递送系统。在压力与健康状态下,EVs释放的分子不同,这些活性分子能够识别机体的患病状态。研究表明,可以利用EVs将药物输送到难以到达的器官,如耳蜗感觉毛细胞和大脑,因为它们能够穿过血迷路和血脑屏障。在本综述中,我们总结了有关EVs的生物组成和发生、分离技术、表征手段、作为药物输送载体的潜在治疗应用、药物加载方法及在听觉系统内耳治疗的应用潜力。     

Flagellin-deficient outer membrane vesicles as adjuvant induce cross-protection of Salmonella Typhimurium outer membrane proteins against infection by heterologous Salmonella serotypes

Salmonella enteric serovar infections result in high morbidity and mortality worldwide. Cross-protective vaccines are an effective strategy in controlling salmonellosis caused by multiple serotypes. In our previous study, outer membrane vesicles (OMVs) derived from flagellin-deficient Salmonella Typhimurium (S. Typhimurium) were proven effective in mediating cross-protection against infection by multiple Salmonella serotypes; OMVs also exhibit potent adjuvant effects. In this study, we further investigated the adjuvant capacities of flagellin-deficient S. Typhimurium OMVs. Our finding showed that outer membrane proteins (OMPs) in combination with flagellin-deficient S. Typhimurium OMVs could function as adjuvants and invoke stronger humoral, cellular, mucosal, and cross-protective immune responses compared to conventional aluminum (alum). Furthermore, as an adjuvant, OMVs could induce significantly higher cellular immune responses and display enhanced cross-protection for OMPs against wild-type virulent Salmonella Choleraesuis and Salmonella Enteritidis challenge. In summary, OMVs function as a potent adjuvant with the capability of conferring greater cross-protection against infection by multiple Salmonella serotypes, and may be of great value as an effective vaccine adjuvant in enteric diseases.     

sncRNAs packaged by Helicobacter pylori outer membrane vesicles attenuate IL-8 secretion in human cells

Bacterial outer membrane vesicles (OMVs) play a vital role in the mechanism of host―pathogen communication, while emerging evidence suggests that OMVs regulate host immune responses through differentially packaged small noncoding RNAs (sncRNAs) to target host mRNA function. Therefore, we identified differentially packaged sncRNAs in Helicobacter pylori OMVs and showed transfer of OMV sncRNAs to human gastric adenocarcinoma cells in this study. Our data revealed that sncRNAs (sR-2509025 and sR-989262) were enriched in OMVs, and reduced lipopolysaccharide or OMV-induced interleukin 8 (IL-8) secretion by cultured AGS cells. Collectively, these findings are consistent with the hypothesis that sncRNAs in H. pylori OMVs play a novel role in the mechanism of host―pathogen interaction, whereby H. pylori evades the host immune response.     

Affinity purification of bacterial outer membrane vesicles (OMVs) utilizing a His-tag mutant

To facilitate the rapid purification of bacterial outer membrane vesicles (OMVs), we developed two plasmid constructs that utilize a truncated, transmembrane protein to present an exterior histidine repeat sequence. We chose OmpA, a highly abundant porin protein, as the protein scaffold and utilized the lac promoter to allow for inducible control of the epitope-presenting construct. OMVs containing mutant OmpA-His6 were purified directly from Escherichia coli culture media on an immobilized metal affinity chromatography (IMAC) Ni-NTA resin. This enabling technology can be combined with other molecular tools directed at OMV packaging to facilitate the separation of modified/cargo-loaded OMV from their wt counterparts. In addition to numerous applications in the pharmaceutical and environmental remediation industries, this technology can be utilized to enhance basic research capabilities in the area of elucidating endogenous OMV function.     

Recombinant N-terminal outer membrane porin (OprF) of Pseudomonas aeruginosa is a promising vaccine candidate against both P. aeruginosa and some strains of Acinetobacter baumannii

Pseudomonas aeruginosa is an evolving pathogen which can cause serious infections especially to immunocompromised patients. Its high resistance profile to antibiotics results in difficulty, and sometimes impossibility, in treating afflicted patients. Developing an effective vaccine against P. aeruginosa is an important approach to tackle this problem. A similar problematic situation exists for Acinetobacter baumannii. Several vaccine candidates have been investigated up till now but still there is no approved vaccine in the market. One important antigen of P. aeruginosa is the outer membrane protein F (OprF) which functions as a porin with relevant important roles in virulence. Previous studies focused mainly on the C-terminal peptidoglycan binding domain of OprF as a vaccine candidate. In the current study, we have investigated the N-terminal porin domain of OprF as a potential vaccine candidate against P. aeruginosa. Histidine-tagged recombinant N-terminal OprF (amino acid range 25–200; OprF_25-200) was overexpressed in Escherichia coli and purified using metal affinity chromatography. Swiss albino mice were immunized with OprF_25-200 adjuvanted with Bacillus Calmette–Guérin (BCG) and alum and the immune response was evaluated. Immunized mice developed antigen-specific IgG1 and IgG2a and were protected against challenge by both P. aeruginosa and a clinical isolate of A. baumannii expressing OprF. Serum from OprF_25-200-immunized mice showed cross-reactivity with both pathogens using western blotting and whole cell enzyme-linked immunosorbent assay (ELISA). To our knowledge, this is the first report to demonstrate that the N-terminal domain of OprF is sufficiently immunogenic to protect against the two pathogens.     

Development of a novel trivalent invasive non-typhoidal Salmonella outer membrane vesicles based vaccine against salmonellosis and fowl typhoid in chickens

Poultry animals act as natural reservoirs of invasive non-typhoidal Salmonella [iNTS] serovars and consumption of iNTS contaminated poultry meat and eggs is one of the major sources of iNTS infection in developed and developing countries. Irrational use of antibiotics in the poultry industry gives rise to the global emergence of multi drug resistant iNTS strains. Among different strategies to control iNTS infection in poultry farms, vaccination is now being widely used. There are several licensed vaccines available in the market for poultry animals to ameliorate iNTS infection but none of them have broad spectrum protective efficacy. In this study we have formulated a single novel trivalent iNTS outer membrane vesicles [OMVs] based immunogen which can confer long term broad spectrum protection against most prevalent iNTS serovars. We have isolated OMVs from Salmonella Typhimurium [ST], Salmonella Enteritidis [SE], and Salmonella Gallinarum [SG] and formulated the trivalent immunogen by mixing OMVs in a 1:1:1 ratio. One day old chicks were immunized thrice via oral route at two week intervals. Vaccination significantly induced serovar specific antibodies detected up to 180 days post immunization. Post challenge with both homologous and heterologous [S. Infantis] serovars, immunized birds showed reduced level of fecal shedding and organ invasion. A long term efficacy study also showed reduced levels of tissue invasion up to one year post immunization. These results demonstrate that our novel formulation of immunogen could be a broad spectrum potential vaccine for both layer and broiler breeds against iNTS mediated salmonellosis and fowl typhoid.     

Surface architecture of Neisseria meningitidis capsule and outer membrane as revealed by atomic force microscopy

An extensive morphological analysis of the Neisseria meningitidis cell envelope, including serogroup B capsule and outer membrane, based on atomic force microscopy (AFM) together with mechanical characterization by force spectroscopic measurements, has been carried out. Three meningococcal strains were used: the encapsulated serogroup B strain B1940, and the isogenic mutants B1940 siaD(+C) (lacking capsule), and B1940 cps (lacking both capsule and lipooligosaccharide outer core). AFM experiments with the encapsulated strain B1940 provided unprecedented images of the meningococcal capsule, which seems to be characterized by protrusions (“bumps”) with the lateral dimensions of about 30 nm. Measurement of the Young's modulus provided quantitative assessment of the property of the capsule to confer resistance to mechanical stress. Moreover, Raman spectroscopy gave a fingerprint by which it was possible to identify the specific molecular species of the three strains analyzed, and to highlight major differences between them.     

沙门菌属外膜蛋白的抗原同源性分析

目的 分析沙门菌属外膜蛋白（outer membrane protein，OMP）的抗原同源性。方法 十二烷基肌氨酸钠法提取甲型副伤寒沙门菌、伤寒沙门菌、鼠伤寒沙门菌、大肠埃希菌、福氏志贺菌、肺炎克雷伯菌、阴沟肠杆菌和粘质沙雷菌的OMP。用甲型副伤寒沙门菌的OMP与完全佐剂制成乳油状，多次多部位免疫日本大耳兔，获得抗OMP抗体血清。35％饱和硫酸铵沉淀并纯化IgG，用HRP标记。SDS-PAGE法分离检测上述8种细菌的OMP。Western blot测定兔抗甲型副伤寒沙门菌OMP抗体与上述其他细菌OMP抗原的反应特异性。斑点印迹．酶联免疫吸附试验（Dot blot-ELISA）测定兔抗甲型副伤寒沙门菌OMP抗体与肠炎沙门菌、费氏枸橼酸杆菌、变形杆菌、铜绿假单胞菌、蜂房哈夫尼亚菌、棒状杆菌、嗜麦芽窄食假单胞菌、金黄色葡萄球菌、摩根摩根菌、屎链球菌、肺炎链球菌、新型隐球菌、热带念珠菌、光滑念珠菌和白色念珠菌提取物的反应性。结果 沙门菌属成员的OMP可与兔抗甲型副伤寒沙门菌OMP抗体血清发生特异性反应；其他革兰阴性菌OMP与之无反应；革兰阳性菌和真菌亦不发生反应。结论 沙门菌属OMP具有高度的抗原同源性，有别于其他细菌。     

An unique method for determining the permeability of the mitochondrial outer membrane

Mitochondrial function depends on the metabolite fluxes through the mitochondrial outer membrane. To directly measure the permeability of the outer membrane by using traditional methods is difficult due to the small volume of the intermembrane space and high permeability of the outer membrane. A method was developed to measure the permeability of the mitochondrial outer membrane by taking advantage of mitochondrial processes that consume the substrate being examined. A steady state concentration of substrate forms in the intermembrane space when the flux through the outer membrane equals the rate of substrate consumption. Thus, the rate of consumption of substrate depends on both processes and the extent of the contribution of each was sorted out by fitting to a steady-state theory. This report focuses on mitochondrial ATP phosphorylation and the determination of the permeability of the outer membrane to ADP. This approach also yields the intermembrane space [ADP] and shows how it differs from the extramitochondrial concentration.