外泌体定量技术研究

正在上世纪80年代Trams等~([1])最早于培养的绵羊红细胞上清液中发现外泌体(exosomes,ES),并首先提出了ES概念。ES是由细胞主动分泌的直径在40~100 nm的囊泡结构~([2]),由磷脂双分子层包裹了细胞质组成,它包含亲代细胞的蛋白质、DNA、RNA、小分子等。近年来,研究人员陆续观察了不同类型细胞中ES的形成过程,并且能够从血浆、尿     

外泌体microRNAs在恶性血液系统疾病中的研究进展

<正>外泌体(exosome)是一类脂质双分子层结构的封闭性膜性囊泡,直径约为30~150 nm~([1]),外泌体的形成最初是由于细胞内吞,细胞膜内化产生核内体,随后在核内体膜的入鞘部分形成许多小囊泡,这样的核内体称之为多泡体,最后多泡体与细胞膜融合,并释放核内体小囊泡到细胞外间隙成为外泌体~([2-3])。外泌体最早是在1983年由Pan和Johnstone发现~([4]),由细胞主动分泌、释放。研究至今,已发现淋巴细胞、血小板、内皮细胞、肥大细     

白血病细胞源外泌体的作用研究进展

<正>白血病(leukemia)是一类造血干/祖细胞恶性克隆性疾病。外泌体是活细胞分泌到胞外的一种纳米级的微囊泡,是细胞间对话的信息和物质载体~([1])。外泌体携带亲本细胞来源的蛋白质、核酸(mRNA、microRNA和DNA等)及脂质等生物信息分子,可近距离和/或经体液流动远距离、特异性调控靶细胞的生理和病理活动~([2-3])。靶细胞摄取外泌体的途径主要有网格蛋白介导的内吞途径、小窝蛋白依赖型内     

外泌体的免疫调节作用及在自身免疫性疾病中的研究

正1外泌体的概述外泌体(Exosomes)是直径大约在30~100 nm之间的双层脂质膜囊泡,可由不同类型的细胞脱落释放,存在于多种体液中,包括血液、尿液、支气管肺泡灌洗液,甚至在唾液和乳汁中也可发现外泌体。与它的起源细胞相比,外泌体含有丰富的胆固醇、神经酰胺、鞘磷脂等脂类成分,这些成分使得外泌体的结构更加稳定~([1])。外泌体包括丰富的蛋白质,如:黏附分子、膜转运因子、趋化因子、信号转导因子、细胞骨架蛋白、热休克蛋白、蛋白酶和细胞特异性抗原     

外泌体参与中枢胶质细胞-神经元之间信息交流和疾病发生的研究进展

正外泌体(exosome)是机体细胞释放到细胞外直径约30~100 nm大小的双层磷脂膜囊泡,其内含有核酸、蛋白质等多种生物活性分子。外泌体参与机体的生理和病理进程,目前被认为是细胞间远程物质传递和信息交流的一种重要方式~([1-3])。Wolf于1967年首次发现血小板来源的囊泡样外泌     

胞外囊泡在肿瘤发生发展中的研究进展

<正>胞外囊泡(extracellular vesicles,EVs)是释放到细胞外的膜性小囊泡,是传递细胞间信号的一种新方式,在生理或病理情况下发挥重要调节作用。几乎所有类型的细胞均可以产生并释放EVs,尤其是肿瘤细胞~([1-2])。越来越多的证据表明,在肿瘤的发生和发展过程中,肿瘤来源的胞外囊泡能够通过传递囊泡中的内容物来改变受体细胞的生物学功能,比如导致免疫抑制,诱导血管生成,甚至促进肿瘤转移~([3-4])。     

外泌体的功能浅析

外泌体是一种由多种细胞分泌,直径在40~100 nm左右的微小囊泡,其间包含了丰富的蛋白质, miRNA以及RNA片段,能够参与细胞之间的物质转导和信号交流。本文将从外泌体的形成、分泌、构成,以及与肿瘤,心血管疾病,神经系统疾病的关系等方面进行综述。     

外泌体标记方法的研究现状

外泌体是细胞分泌的直径为30～150 nm的胞外囊泡,可以运送多种生物活性分子(如RNA、脂质和蛋白质),介导细胞间的信息交流。现已发现,外泌体在各种生理和病理过程中发挥着重要的作用。为了研究外泌体的生物学作用,现已开发出了多种外泌体标记方法进行外泌体的功能研究。根据现有的文献报道,文章将结合外泌体自身的结构特点,对不同的外泌体标记方法,包括荧光染料、荧光蛋白、荧光素酶和物理标记等作一综述。     

RNA结合蛋白HuR对外泌体生成机制的调控

外泌体是一类直径为30～150 nm的膜囊泡,几乎所有的细胞都能够分泌外泌体,它含有许多来源于细胞的成分.近年来,越来越多的证据表明外泌体介导的信号蛋白、RNA、核酸等多种分子的传递有助于肿瘤的发生发展,包括重塑细胞外基质、促进血管生成、干扰免疫反应、促进肿瘤细胞的增殖迁移以及塑造肿瘤转移前微环境等[1].     

外泌体作为药物递送系统的研究进展

外泌体是一种由细胞分泌的直径约40-100 nm的盘状囊泡,具有天然脂质双分子层,其内包含了复杂的RNA和蛋白质.大多数细胞在正常及病理状态下均能产生外泌体,这些外泌体主要存在于体液中,在细胞间物质运输和信号交流中发挥着重要作用.作为天然内源性纳米级载体,外泌体具有毒性小、稳定性好、、渗透性好、靶向归巢性强、能透过血脑...     

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.     

人脂肪间充质干细胞来源的外排体促进大鼠创伤性脑损伤后神经功能恢复

目的研究人脂肪间充质干细胞(h AMSCs)来源的外排体对创伤性脑损伤(TBI)的治疗作用及其可能的机制。方法分离健康成人脂肪MSCs,通过超滤法提取外排体。将大鼠分成:假手术组,PBS对照组,MSC治疗组,exosomes治疗组。于TBI建模24 h后,治疗组分别沿损伤边缘区局部注射,PBS 30μL,MSC 2×10~5个细胞/只,exosomes 25μg总蛋白量/只,总体积30μL。在建模前和TBI后1、3、7、10、13、16和30 d测试所有大鼠的m NSS评分和前肢踩空试验。3和7d处死大鼠,提取大鼠脑组织总RNA,实时定量PCR检测大鼠炎性因子TNF-α和IL-1β的表达,30 d处死大鼠,tunel-neun双标免疫荧光检测TBI后神经元凋亡。结果外排体的治疗显著促进TBI后的神经功能的恢复,治疗效果与MSC治疗效果相当,其机制可能是通过抑制大鼠TBI后急性炎性反应,减少神经元凋亡。结论人脂肪间充质干细胞来源的外排体促进脑外伤后神经功能的恢复,这将为临床提供一种新的更安全的TBI治疗手段。     

间充质干细胞外泌体在神经系统疾病修复过程中的作用与应用

文题释义：外泌体：是直径在30-120 nm的封闭脂质囊泡,属于多泡体分泌的细胞外囊泡。多种细胞在生理或病理情况下都能分泌外泌体。外泌体作为细胞间重要信息交流工具,在新型组织修复、疾病治疗与诊断领域有重要的前景。 间充质干细胞：是中胚层的一种多能干细胞,具有自我更新、向多种间充质系列细胞(如成骨、成软骨及成脂肪细胞等)或非间充质系列细胞分化、分泌细胞因子和细胞外囊泡、免疫调节、来源广泛等特点,被广泛用于组织修复研究。 背景：由于血脑屏障的存在,大分子药物无法通过血脑屏障进入脑部组织发挥药效,导致很多神经系统疾病、神经退行性疾病无法得到有效的治疗。近年来研究发现间充质干细胞外泌体以其体积微小、可装载脂质、蛋白、核酸等信号物质的特点,对脑血管病、阿尔茨海默症、癫痫、脊髓损伤等疾病具有组织修复的功效,逐渐成为治疗神经系统疾病的重要工具。 目的：从宏观与微观角度对间充质干细胞外泌体在神经系统疾病修复过程中的作用进行分析总结,并提出在外泌体基础研究和临床试验中存在的问题和注意事项。 方法：以“exosomes,extracellular vesicles,MSCs,mesenchymal stem cells,neurodegenerative diseases”为英文检索词,通过计算机检索PubMed数据库,纳入描述间充质干细胞来源外泌体的特性及修复作用的文章,排除重复与不相关文章,最终整理出35篇文献进行综述。 结果与结论：间充质干细胞外泌体具有易穿过血脑屏障、携带丰富的信号物质等生物学特点,在动物疾病模型中发挥重要作用,如抗炎、促进神经元生长、维持神经元数量、促进神经突重塑等;经过修饰后的外泌体可发挥比天然外泌体更有效的组织修复功能,可作为分子药物载体应用于特定的神经系统疾病治疗中。 ORCID: 0000-0002-1791-666X(高振橙) 中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

The voltage-gated proton channel Hv1 enhances brain damage from ischemic stroke

Phagocytic cell NADPH oxidase (NOX) generates reactive oxygen species (ROS) as part of innate immunity. Unfortunately, ischemia can also induce this pathway and inflict damage on native cells. The voltage-gated proton channel Hv1 enables NOX function by compensating cellular loss of electrons with protons. Accordingly, we investigated whether NOX-mediated brain damage in stroke can be inhibited by suppression of Hv1. We found that mouse and human brain microglia, but not neurons or astrocytes, expressed large Hv1-mediated currents. Hv1 was required for NOX-dependent ROS generation in brain microglia in situ and in vivo. Mice lacking Hv1 were protected from NOX-mediated neuronal death and brain damage 24 h after stroke. These results indicate that Hv1-dependent ROS production is responsible for a substantial fraction of brain damage at early time points after ischemic stroke and provide a rationale for Hv1 as a therapeutic target for the treatment of ischemic stroke.     

New insights into mechanism for the effect of resveratrol preconditioning against cerebral ischemic stroke: Possible role of matrix metalloprotease-9

Resveratrol, a natural polyphenolic compound, is found in a few edible materials and is well known for its phytoestrogenic and antioxidant properties. A growing body of in vivo and in vitro evidence indicates that resveratrol has protective effect on cerebral ischemic stroke. Here, we review the effect of resveratrol on cerebral ischemic stroke, and propose a possible mechanism. During acute phases after stroke, resveratrol preconditioning suppresses matrix metalloprotease-9 activity to ameliorate blood-brain barrier disruption, edema formation and neuronal cell death caused by ischemia and reperfusion. But during delayed phases after stroke, resveratrol preconditioning conduces to cerebral angiogenesis and brain regeneration through increasing matrix metalloprotease-9 activity and expression. Resveratrol's effect on matrix metalloprotease-9 is distinguishing in different phases because of temporal and spatial redistribution of matrix metalloprotease-9 within the cells of the neurovascular unit after cerebral ischemia. This paper also hypothesizes that resveratrol treatment after cerebral ischemia might be beneficial for cerebral angiogenesis and brain regeneration during delayed phases after stroke.     

Reactive oxygen radicals and pathogenesis of neuronal death after cerebral ischemia

Reactive oxygen species have been implicated in brain injury after cerebral ischemia. These oxidants can damage proteins, lipids, and DNA, and lead to cell injury and necrosis. Oxidants are also initiators in intracellular cell death signaling pathways that may lead to apoptosis. The possible targets of this redox signaling include mitochondria, death membrane receptors, and DNA repair enzymes. Genetic manipulation of intrinsic antioxidants and the factors in the signaling pathways has provided substantial progress in understanding the mechanisms in cell death signaling pathways and involvement of oxygen radicals in ischemic brain injury. Future studies of these pathways may provide novel therapeutic strategies in clinical stroke.     

Exosome-mediated transfer of miR-133b from multipotent mesenchymal stromal cells to neural cells contributes to neurite outgrowth

Multipotent mesenchymal stromal cells (MSCs) have potential therapeutic benefit for the treatment of neurological diseases and injury. MSCs interact with and alter brain parenchymal cells by direct cell-cell communication and/or by indirect secretion of factors and thereby promote functional recovery. In this study, we found that MSC treatment of rats subjected to middle cerebral artery occlusion (MCAo) significantly increased microRNA 133b (miR-133b) level in the ipsilateral hemisphere. In vitro, miR-133b levels in MSCs and in their exosomes increased after MSCs were exposed to ipsilateral ischemic tissue extracts from rats subjected to MCAo. miR-133b levels were also increased in primary cultured neurons and astrocytes treated with the exosome-enriched fractions released from these MSCs. Knockdown of miR-133b in MSCs confirmed that the increased miR-133b level in astrocytes is attributed to their transfer from MSCs. Further verification of this exosome-mediated intercellular communication was performed using a cel-miR-67 luciferase reporter system and an MSC-astrocyte coculture model. Cel-miR-67 in MSCs was transferred to astrocytes via exosomes between 50 and 100 nm in diameter. Our data suggest that the cel-miR-67 released from MSCs was primarily contained in exosomes. A gap junction intercellular communication inhibitor arrested the exosomal microRNA communication by inhibiting exosome release. Cultured neurons treated with exosome-enriched fractions from MSCs exposed to 72 hours post-MCAo brain extracts significantly increased the neurite branch number and total neurite length. This study provides the first demonstration that MSCs communicate with brain parenchymal cells and may regulate neurite outgrowth by transfer of miR-133b to neural cells via exosomes.     

Immune responses in stroke: how the immune system contributes to damage and healing after stroke and how this knowledge could be translated to better cures?

Stroke is one of the leading causes of death and disability worldwide. The long‐standing dogma that stroke is exclusively a vascular disease has been questioned by extensive clinical findings of immune factors that are associated mostly with inflammation after stroke. These have been confirmed in preclinical studies using experimental animal models. It is now accepted that inflammation and immune mediators are critical in acute and long‐term neuronal tissue damage and healing following thrombotic and ischaemic stroke. Despite mounting information delineating the role of the immune system in stroke, the mechanisms of how inflammatory cells and their mediators are involved in stroke‐induced neuroinflammation are still not fully understood. Currently, there is no available treatment for targeting the acute immune response that develops in the brain during cerebral ischaemia. No new treatment has been introduced to stroke therapy since the discovery of tissue plasminogen activator therapy in 1996. Here, we review current knowledge of the immunity of stroke and identify critical gaps that hinder current therapies. We will discuss advances in the understanding of the complex innate and adaptive immune responses in stroke; mechanisms of immune cell‐mediated and factor‐mediated vascular and tissue injury; immunity‐induced tissue repair; and the importance of modulating immunity in stroke.     

Exosomes Mediate the Intercellular Communication after Myocardial Infarction

The mechanisms of cardiac repair after myocardial infarction (MI) are complicated and not well-understood currently. It is known that exosomes are released from most cells, recognized as new candidates with important roles in intercellular and tissue-level communication. Cells can package proteins and RNA messages into exosome and secret to recipient cells, which regulate gene expression in recipient cells. The research on exosomes in cardiovascular disease is just emerging. It is well-known that exosomes from cardiomyocyte can transfect endothelial cells, stem cells, fibroblasts and smooth muscle cells to induce cellular changes. After myocardial infarction (MI), the exosomes play important roles in local and distant microcommunication. Nowadays, exosomal microRNAs transportation has been found to deliver signals to mediate cardiac repair after MI. However, the exosomes quality and quantities are variable under different pathological conditions. Therefore, we speculate that the monitoring of the quality and quantity of exosomes may serve as diagnosis and prognosis biomarkers of MI, and the study of exosomes will provide insights for the new therapeutics to cardiac remodeling after MI.     

Intracerebral injection of autologous whole blood in rats: time course of inflammation and cell death

Intracerebral hemorrhage is associated with stroke and head trauma. The purpose of this study was to study brain inflammation and cell death in adult rats 1 h to 4 weeks after injection of blood into the striatum. Terminal dUTP nick-end-labeling positive dying cells were evident 4 h to 4 weeks post-hemorrhage. Neutrophil infiltration was brief and peaked at 48 h. CD8a immunoreactive lymphocytes, possibly natural killer cells, became apparent at 48 h and persisted for 1 week. Microglial reaction was evident at 4 h and persisted for 4 weeks. We conclude that extravascular blood causes a mixed inflammatory cell reaction in brains that is maximal from 48-72 h following hemorrhage. This is associated with death of brain cells over a prolonged period of at least 4 weeks.