树突状细胞及exosomes体外诱导抗脑胶质瘤细胞毒活性的研究

目的探讨负载肿瘤抗原的树突状细胞（DCs）及其衍生exosomes诱导T细胞活化和特异性肿瘤杀伤的作用。方法将患者静脉血单个核细胞经GM—CSF、IL-4诱导产生DCs,收集培养上清液,超速离心法制备exosomes。流式细胞术检测DCs表面标志表达,用SDS-PAGE和western—blot检测exosomes携带MHC和共刺激分子的情况。MI]r法观察DCs及exosomes对T细胞促增殖作用和CTL体外杀伤活性。结果肿瘤抗原致敏的DC高表达MHC—I、MHC-Ⅱ、CD54、CDS0和CD86,与抗原致敏前相比有显著差异,P〈0．05。DC衍生的exosomes表达MHC-Ⅱ、CD54和CD86,并显著引起T细胞扩增和细胞毒效应,与对照组相比有显著差异,P〈0．01。结论从脑胶质瘤患者静脉血诱导出高表达MHC和共刺激分子的DCs以及exosomes,具有活化T细胞并产生特异性细胞毒活性的功能,有望成为临床治疗脑胶质瘤有效的新疫苗。     

DC-SIGN:C型凝集素中的新成员

DC SIGN是一种树突状细胞特征性的且具有多种功能免疫新分子 ,不但可以参与树突状细胞的迁移、粘附以及对抗原的识别与内化 ,还可以参与许多病原体的免疫逃避 ,本文拟就有关内容作一综述。     

DC-SIGN：C型凝集素中的新成员

DC-SIGN是一种树突状细胞特征性的且具有多种功能免疫新分子，不但可以参与树突状细胞的迁移、粘附以及对抗原的识别与内化，还可以参与许多病原体的免疫逃避，本文拟就有关内容作一综述。     

肿瘤外泌体对免疫细胞的作用

外泌体是细胞主动分泌的双层囊泡小体,广泛存在于各种体液中,直径通常为30～100 nm。外泌体能将其携带的的脂类、蛋白质、核酸等多种重要的生物功能分子运输到受体细胞中,起到物质传递与信息交流的作用。肿瘤来源外泌体(tumor-derived exosomes,TEXs)与肿瘤的发生发展密切相关,既可作为抗原引发抗肿瘤免疫,更重要的是,也可负向调控多种免疫细胞功能,帮助肿瘤细胞免疫逃逸。本文主要对外泌体在多种肿瘤中对各类免疫细胞的调控作用进行分析总结。     

肿瘤微环境中髓系来源抑制细胞对T细胞免疫活性抑制的分子机制

髓系来源抑制细胞(MDSC)根据其起源和功能而命名,由巨噬细胞、树突状细胞(DC)及粒细胞等细胞的前体细胞组成。肿瘤免疫逃逸与肿瘤微环境中MDSC介导的抗肿瘤免疫抑制相关,肿瘤微环境中的MDSC主要通过抑制T细胞的免疫活性来发挥免疫抑制作用。根据MDSC与T细胞之间的接触方式,肿瘤微环境中的MDSC抑制T细胞免疫功能的分子机制可分为直接抑制和间接抑制两类,其中MDSC通过产生细胞因子影响T细胞功能和细胞之间膜受体配体相互作用发挥抑制T细胞功能的方式为直接抑制,而MDSC通过影响T细胞代谢从而影响T细胞功能及通过其他细胞发挥抑制T细胞功能的方式为间接抑制。制定针对MDSC的治疗策略时,需综合考虑上述多种分子机制共存的可能性。     

Lewis大鼠骨髓来源成熟和未成熟树突状细胞的提取与鉴别

背景：树突状细胞在未成熟阶段表现出极强的抗原吞噬功能，它可以在免疫耐受、癌症的免疫治疗等方面都表现出极大的优越性。但由于未成熟树突状细胞在生物体内含量极微，这就严重限制了它在临床、科研方面的应用。目的：提取鉴别Lewis大鼠骨髓来源成熟和未成熟树突状细胞。方法：从Lewis大鼠骨髓中分离骨髓前体细胞，应用20 ng/m L粒细胞集落刺激因子、10 ng/m L白细胞介素4培养7 d诱导为未成熟树突状细胞，然后在未成熟树突状细胞中加入1μg/m L脂多糖继续培养2 d诱导为成熟树突状细胞。采用荧光倒置显微镜观察树突状细胞形态，流式细胞仪鉴定成熟和未成熟树突状细胞表面特异性分子，ELISA检测成熟和未成熟树突状细胞培养上清白细胞介素10、白细胞介素12和白细胞介素17A因子的分泌水平，混合淋巴细胞反应检测成熟和未成熟树突状细胞对T淋巴细胞的刺激反应。结果与结论：(1)普通荧光倒置显微镜下观察树突状细胞具有明显的突起结构；(2)流式细胞仪可见未成熟树突状细胞低表达CD40、CD86等共刺激分子；相反，成熟树突状细胞高表达上述共刺激分子；(3)未成熟树突状细胞的白细胞介素10、白细胞介素17A...     

外泌体在肿瘤微环境中的免疫抑制作用

外泌体(exosome)作为一种纳米级囊泡,在细胞间的信息传递中起着重要作用。越来越多的研究表明,肿瘤细胞来源外泌体(tumor-derived exosomes,TEXs)携带有与亲代肿瘤细胞相似的免疫抑制性分子,介导了肿瘤细胞与NK细胞、树突状细胞、髓源性抑制细胞、巨噬细胞、效应性T细胞、调节性T细胞和调节性B细胞的通讯,从而抑制肿瘤微环境中的抗肿瘤免疫应答。此外,近年来研究发现免疫细胞来源的外泌体在免疫抑制性肿瘤微环境的形成过程中也起一定作用。现针对外泌体的生物学特征及其在免疫抑制性肿瘤微环境形成过程中的作用进行综述。     

热休克E.G7-OVA肿瘤细胞来源的exosomes的抗肿瘤作用

目的 本研究探讨热休克E.G7-OVA肿瘤细胞来源的exosomes的体内抗肿瘤效应.方法 通过分级离心和蔗糖密度梯度离心法分离和纯化E.G7-OVA肿瘤细胞来源的exosomes.热休克E.G7-OVA肿瘤细胞来源的exosomes和未热休克E.G7-OVA肿瘤细胞来源的exosomes分别命名为Exo/HS和Exo.通过电镜观察exosomes的形态,Western blot检测exosomes的蛋白成分.以Exo、Exo/HS、PBS免疫小鼠,用E.G7-OVA肿瘤细胞进行攻击,观察各组免疫保护效应;建立E.G7-OVA荷瘤小鼠模型,观察各组免疫治疗效应.通过LDH法检测各组免疫小鼠脾细胞CTL活性.结果 电镜下exosomes为双层膜的囊泡样结构,直径为40～100 nm.Western blot结果 表明:Exo和Exo/HS都含有HSC70、HSP70、HSP60、HSP90、MHC Ⅰ和OVA分子,而Exo/HS中HSP70和MHC Ⅰ分子的含量更高.免疫保护试验发现,Exo/HS组免疫小鼠90 d的无瘤率显著高于Exo组和PBS组(50%、20%、0%,P<0.01);对荷瘤小鼠的免疫治疗显示,Exo/HS对荷瘤小鼠的肿瘤抑制效应显著高于Exo组和PBS组(P<0.01).CTL结果 表明,Exo/HS免疫小鼠诱导的OVA抗原特异性的CTL活性显著高于Exo组和PBS组(P<0.01).结论 热休克E.G7-OVA肿瘤细胞来源的exosomes可作为有效的肿瘤疫苗.     

树突状细胞与特异性细胞免疫

树突状细胞是目前发现的递呈功能最强的抗原递呈细胞,它在抗原摄取与递呈方面的独特作用越来越引起人们的重视;同时,它通过提供双信号刺激、细胞辅助作用、细胞因子等直接和间接地启动特异性细胞免疫。在免疫应答中发挥着极其重要的作用。     

聚肌胞苷酸对人胎盘胎儿源性间充质干细胞细胞因子产生的影响

<正>胎盘来源的间充质干细胞(plancenta derived mesenchymal stem cells,PMSC)越来越多的用于临床治疗[1]。PMSC免疫调节功能可能与抑制树突状细胞、T细胞、B细胞等多种免疫细胞功能,同时释放多种免疫调控因子相关[2]。其中一些免疫调控因子的分泌受到Toll样受体(Toll-like receptor,TLR)信号通路的调控。因此TLR激活剂可能诱导人胎盘胎儿     

Dendritic cell derived-exosomes: biology and clinical implementations

Exosomes are nanometer-sized membrane vesicles invaginating from multivesicular bodies and secreted from different cell types. They represent an "in vitro" discovery, but vesicles with the hallmarks of exosomes are present in vivo in germinal centers and biological fluids. Their protein and lipid composition is unique and could account for their expanding functions such as eradication of obsolete proteins, antigen presentation, or "Trojan horses" for viruses or prions. The potential of dendritic cell-derived exosomes (Dex) as cell-free cancer vaccines is addressed in this review. Lessons learned from the pioneering clinical trials allowed reassessment of the priming capacities of Dex in preclinical models, optimizing clinical protocols, and delineating novel, biological features of Dex in cancer patients.     

Nanovesicular vaccines: exosomes

Exosomes are small membrane vesicles derived from late endosome. They are about 30--100 nm in diameter. The secretion of exosomes is a process in which multivesicular bodies fuse with the cell membrane, and all cells that contain multivesicular endocytic compartments could theoretically secrete exosomes. The surprising biological functions of exosomes are only slowly being unveiled, but it is already clear that they serve to remove obsolete membrane proteins and act as messages of inter-cellular communication. Exosomes derived from tumor or antigen-presenting cells have been extensively investigated. They are released into the extracellular environment and fuse with the membranes of neighboring cells, delivering membrane and cytoplasmic proteins from one cell to another. Exosomes carry immunorelevant structures which play important roles in immune response, such as MHC molecules, costimulatory molecules, heat shock proteins, and naive tumor antigens. Therefore they have been suggested as potential vaccines. Consequently, exosomes have shown considerable anti-tumor effect in several studies and are in phase I clinical trials.     

REVIEW ARTICLE: The Role of Placental Exosomes in Reproduction

Citation Mincheva‐Nilsson L, Baranov V. The Role of Placental Exosomes in Reproduction. Am J Reprod Immunol 2010 Cell communication comprises cell–cell contact, soluble mediators and intercellular nanotubes. There is, however, another cell–cell communication by released membrane‐bound microvesicles that convey cell–cell contact ‘by proxy’ transporting signals/packages of information from donor to recipient cells locally and/or at a distance. The nanosized exosomes comprise a specialized type of microvesicles generated within multivesicular bodies (MVB) and released upon MVB fusion with the plasma membrane. Exosomes are produced by a variety of immune, epithelial and tumor cells. Upon contact, exosomes transfer molecules that can render new properties and/or reprogram their recipient cells. Recently, it was discovered that the syncytiotrophoblast constitutively and throughout the pregnancy secretes exosomes. The placenta‐derived exosomes are immunosuppressive and carry proteins and RNA molecules that in a redundant way influence a number of mechanisms and promote the fetal allograft survival. In this review, we summarize the current knowledge on the nature of placenta‐derived exosomes and discuss their role in pregnancy.     

调节性DC分泌的外体诱导免疫耐受功能的体外研究

为了探讨树突状细胞(DC)分泌的外体(Dex)在诱导T细胞免疫耐受中的作用,体外研究采用供体Dex降低同种异体移植排斥的可能性。从正常人外周血单个核细胞中诱导培养未成熟DC(imDC),用TGF-β1联合IL-10诱导调节性DC,LPS诱导DC成熟。采用流式细胞术方法观察TGF-β1和IL-10对DC表型、吞噬功能的影响;采用超速离心和超滤的方法提纯Dex;Western blot方法检测imDC分泌的Dex(imDex)与调节性DC分泌的Dex(rDex)表达的相关分子;通过CCK-8法分析异源iDex和mDex在混合淋巴细胞反应(MLR)中的生物学功能,并比较rDex与iDex诱导免疫耐受的能力。结果显示,TGF-β1和IL-10可下调DC表面的共刺激分子CD80、CD83、CD86的表达,并诱导调节性DC分泌更多的rDex;异源的mDC分泌的Dex(mDex)在mDC存在时增强MLR,而异源的imDex在imDC存在时一定程度上抑制MLR,rDex诱导的抑制T细胞增殖作用显著强于iDex;rDex表达更多的FasL,提示TGF-β1和IL-10诱导的调节性DC分泌的rDex在免疫耐受中发挥重要作用,有望应用于同种异体移植抗免疫排斥。     

Exosomes: A Novel Pathway of Local and Distant Intercellular Communication that Facilitates the Growth and Metastasis of Neoplastic Lesions

Normal and diseased cells release bilayered membrane-bound nanovesicles into interstitial spaces and into bodily fluids. A subgroup of such microvesicles is called exosomes and is described in blood as 30 to 100 nm in diameter and as spherical to cup-shaped nanoparticles with specific surface molecular characteristics (eg, expression of the tetraspanins CD9, CD81, and CD63). Extracellular microvesicles provide local signals (eg, autocrine and paracrine) and distant endocrine signals to cells via the transfer of their contents, which include signal proteins, lipids, miRNAs, and functional mRNAs. Exosomes and related microvesicles also aid cells in exporting less-needed molecules and potentially harmful molecules, including drugs; in the case of neoplasia, the export of chemotherapeutic drugs may facilitate cellular chemoresistance. Cancers have adapted the exosome and related microvesicles as a pathway by which neoplastic cells communicate with each other (autocrine) and with nonneoplastic cells (paracrine and endocrine); via this pathway, cancer suppresses the immune system and establishes a fertile local and distant environment to support neoplastic growth, invasion, and metastases. Because exosomes mirror and bind to the cells from which they arise, they can be used for delivery of drugs, vaccines, and gene therapy, as biomarkers and targets. We review how exosomes and related extracellular microvesicles facilitate the progression and metastases of cancers and describe how these microvesicles may affect clinical care.Exosomes are a subcategory of bilayer membrane-bound nanovesicles released from normal and diseased cells into interstitial spaces and, in some cases, into bodily fluids. Exosomes also are released by cultured cells into media. Exosomes are defined and separated from other vesicles based on their source, method of isolation, sizes, and surface markers. Exosomes and other vesicles frequently are unrecognized as to their importance to physiological and pathological processes because they are essentially invisible; their small sizes keep them suspended in fluids so that their effects may not be identified in that their contents and functions would seem to be consistent with those of soluble molecules. Exosomes and related microvesicles were once thought to be artifacts and/or cellular trash, but now exosomes are accepted as a component of a newly identified intercellular communication system that can modulate the functions of target cells. The involvement of exosomes and related microvesicles in providing autocrine (ie, local signals between the same cell type, such as cancer cells), paracrine (ie, local signals between different cell types, such as between epithelial cancer cells and stromal cells), and endocrine (ie, distant signals between any types of cells usually carried in bodily fluids, such as blood) signals has led to the frequent use of the term, signalosomes, being applied to these structures.     

Exosomes in tumor microenvironment influence cancer progression and metastasis

Exosomes are small membrane vesicles of endocytic origin with a size of 50–100 nm. They can contain microRNAs, mRNAs, DNA fragments, and proteins, which are shuttled from a donor cell to recipient cells. Many different cell types including immune cells, mesenchymal cells, and cancer cells release exosomes. There is emerging evidence that cancer-derived exosomes contribute to the recruitment and reprogramming of constituents associated with tumor environment. Here, we discuss different mechanisms associated with biogenesis, payload, and transport of exosomes. We highlight the functional relevance of exosomes in cancer, as related to tumor microenvironment, tumor immunology, angiogenesis, and metastasis. Exosomes may exert an immunosuppressive function as well as trigger an anti-tumor response by presenting tumor antigens to dendritic cells. Exosomes may serve as cancer biomarkers and aid in the treatment of cancer.     

Mesenchymal stem cell-derived extracellular vesicle-based therapies protect against coupled degeneration of the central nervous and vascular systems in stroke

Stem cell-based treatments have been suggested as promising candidates for stroke. Recently, mesenchymal stem cells (MSCs) have been reported as potential therapeutics for a wide range of diseases. In particular, clinical trial studies have suggested MSCs for stroke therapy. The focus of MSC treatments has been directed towards cell replacement. However, recent research has lately highlighted their paracrine actions. The secretion of extracellular vesicles (EVs) is offered to be the main therapeutic mechanism of MSC therapy. However, EV-based treatments may provide a wider therapeutic window compared to tissue plasminogen activator (tPA), the traditional treatment for stroke. Exosomes are nano-sized EVs secreted by most cell types, and can be isolated from conditioned cell media or body fluids such as plasma, urine, and cerebrospinal fluid (CSF). Exosomes apply their effects through targeting their cargos such as microRNAs (miRs), DNAs, messenger RNAs, and proteins at the host cells, which leads to a shift in the behavior of the recipient cells. It has been indicated that exosomes, in particular their functional cargoes, play a significant role in the coupled pathogenesis and recovery of stroke through affecting the neurovascular unit (NVU). Therefore, it seems that exosomes could be utilized as diagnostic and therapeutic tools in stroke treatment. The miRs are small endogenous non-coding RNA molecules which serve as the main functional cargo of exosomes, and apply their effects as epigenetic regulators. These versatile non-coding RNA molecules are involved in various stages of stroke and affect stroke-related factors. Moreover, the involvement of aging-induced changes to specific miRs profile in stroke further highlights the role of miRs. Thus, miRs could be utilized as diagnostic, prognostic, and therapeutic tools in stroke. In this review, we discuss the roles of stem cells, exosomes, and their application in stroke therapy. We also highlight the usage of miRs as a therapeutic choice in stroke therapy.     

CD4+CD25+ Treg 细胞分泌外泌体在疾病中作用的研究进展

调节性T 细胞为T 细胞的一类控制体内自身免疫反应性的T 细胞亚群,在维持机体的免疫耐受以及调控免 疫应答中起重要作用。外泌体是细胞分泌的异质性纳米级胞外囊泡。现在研究认为外泌体在细胞间通讯中发挥重要作用, 外泌体可将其细胞内的多种RNA、DNA 片段、脂质和蛋白质等物质转送到不同的受体细胞,从而改变受体细胞的生物学活性。 多项研究证据表明Treg 细胞可分泌外泌体发挥免疫调节作用,并参与感染免疫、器官移植、超敏反应、自身免疫病以及肿瘤的 发生与发展。本文对Treg 细胞来源外泌体的组成成分、形成途径以及其免疫调节作用等进行综述。     

Dexamethasone inhibits the antigen presentation of dendritic cells in MHC class II pathway

Glucocorticoids (GC) are physiological inhibitors of inflammatory responses and are widely used as anti-inflammatory and immunosuppressive agents in treatment of many autoimmune and allergic diseases. In the present study, we demonstrated that one of the mechanisms by which GC can suppress the immune responses is to inhibit the differentiation and antigen presentation of dendritic cells (DC). DC were differentiated from murine bone marrow hematopoietic progenitor cells by culture with GM-CSF and IL-4 with or without dexamethasone (Dex). Our data showed that Dex, in a dose dependent manner, down-regulated surface expression of CD86, CD40, CD54 and MHC class II molecules by DC, but the expression of MHC class I, CD80, CD95 and CD95L were not affected. In addition, Dex-treated DC showed an impaired function to activate alloreactive T cells and to secrete IL-Ibeta and IL-12p70. Moreover, Dex inhibited DC to present antigen by MHC class II pathway. However, the endocytotic activity of DC was not affected. The inhibitory effect of Dex on the expression of costimulatory molecules and the antigen-presenting capacity of DC could be blocked by the addition of RU486, a potent steroid hormone antagonist, suggesting the requirement of binding to cytosolic receptors in the above-described action of Dex. Since DC have the unique property to present antigen to responding naive T cells and are required in the induction of a primary response, the functional suppression of DC by Dex may be one of the mechanisms by which GC regulate immune responses in vivo.