寒冷刺激支气管上皮细胞产生外泌体促肺成纤维细胞表达气道重塑相关因子北大核心CSCD

外泌体可由多种细胞分泌,是具有多种生物学功能的细胞外囊泡,但其在气道重塑中的作用尚不明确。为探讨经寒冷刺激的人支气管上皮细胞(BEAS-2B)分泌的外泌体对人胚肺成纤维细胞(HLF1)气道重塑相关因子表达的影响,收集BEAS-2B细胞株培养液提取外泌体,利用透射电镜及Western印迹对外泌体进行其大小、形态及标志性蛋白的检测;提取的外泌体与HLF1共同培育,分别设置空白对照组、正常对照组(加入未作干预的BEAS-2B细胞所产的外泌体)及寒冷刺激组(加入经寒冷刺激后的BEAS-2B细胞所产的外泌体)。运用Real-time PCR及Western印迹技术,分别检测各组HLF1表达FGF-2、TNF-α、MMP-9的mRNA及蛋白情况。结果显示,提取BEAS-2B细胞分泌的外泌体为直径小于100 nm的圆形或椭圆形结构,并表达外泌体标志性蛋白CD9、TSG101、ALIX;寒冷刺激组24 h后,其FGF-2、TNF-α、MMP-9的mRNA及蛋白表达均显著高于空白对照组及正常对照组(均P<0.05)。本研究结果表明,BEAS-2B细胞能够释放外泌体;经寒冷刺激后的BEAS-2B细胞所释放的外泌体可以携带并传递生物信号,诱导HLF1表达气道重塑相关因子。     

骨髓间充质干细胞外泌体miR-190a-5p通过靶向KLF15抑制肺癌细胞迁移和侵袭

目的:探讨骨髓间充质干细胞(BMSC)来源的外泌体miR-190a-5p对肺癌细胞的影响。方法:通过超速离心获得BMSCs外泌体,透射电镜观察外泌体形态,采用纳米颗粒示踪分析(NTA)检测外泌体粒径,利用Western印迹检测外泌体上的标志蛋白CD63、CD9及HSP70;选取肺癌细胞系A549、LK79、H1975和HCC827,以及人正常上皮细胞BEAS-2B检测对比miR-190a-5p在这些细胞中和BMSCs衍生的外泌体(BMSC-exosome)中的表达量;双萤光素酶报告基因检测验证Krüppel样因子15(KLF15)是否为miR-190a-5p的靶基因;定量PCR(qRT-PCR)和Western印迹检测miR-190a-5p对KLF15的表达调控;Transwell法检测外泌体对肺癌细胞迁移和侵袭的影响。结果:BMSCs外泌体呈圆形,粒径集中在150～200 nm,标志蛋白CD63、CD9及HSP70阳性表达;BMSCs外泌体中miR-190a-5p的相对表达量均高于在4种肺癌细胞及正常肺细胞BEAS-2B中的表达;双萤光素酶报告基因检测KLF15是miR-190a-5p的靶基因;BMSCs外泌体与miR-190a-5p mimics均能使肺癌细胞中的miR-190a-5p含量升高,并抑制KLF15的mRNA和蛋白表达,从而抑制肺癌细胞迁移和侵袭。结论:BMSCs外泌体miR-190a-5p通过下调KLF15抑制肺癌细胞迁移和侵袭,为肺癌的诊断和治疗提供了新的思路。     

一种大容量细胞灌流液中外泌体的提取方法及鉴定*

目的: 建立一种从大容量细胞灌流液中提取外泌体的方法,并进行外泌体的鉴定。方法: 人脐静脉内皮细胞株(EA.HY926)是人脐静脉内皮细胞和人肺腺癌细胞株A549杂交成的永生化细胞株,因其具有血管内皮细胞的特性,广泛用于内皮细胞相关研究。本研究采用含10%胎牛血清的M199培养基培养,利用Flexcell STR-4000平行板流室系统对EA.HY926施以振荡剪切应力。收集流体剪切应力处理后的细胞灌流液,去除细胞碎片后冻成干粉,脱盐、提取纯化外泌体。电镜观察外泌体形态、纳米粒径电位分析仪检测外泌体大小、PKH26染色检测外泌体膜性结构、BCA蛋白定量法检测外泌体的蛋白浓度、Western blot检测外泌体特异性蛋白CD9和CD81的表达,荧光定量RT-PCR检测内皮细胞相关基因的表达。结果: 该方法提取的外泌体,大小均一,结构完整,呈典型囊泡样结构;粒径集中在30~150 nm,多数粒径为97.63 nm;表达外泌体特异性蛋白CD9和CD81;PKH26染色阳性,并可被细胞摄取;EA.HY926分泌的外泌体表达内皮细胞相关的CD31、vWF等mRNA,以及miR-126、miR-21、miR-155等microRNA分子。结论: 本方法能够有效从大容量细胞灌流液中提取到结构完整的、高浓度、高质量的外泌体,为开展以流体力学干预细胞为基础的外泌体相关研究提供技术方法。     

胶质瘤来源外泌体通过高迁移率族蛋白B1促进胶质瘤干细胞形成

摘要 目的：研究胶质瘤来源外泌体中高迁移率族蛋白B1（HMGB1）对胶质瘤干细胞形成的影响及其意义。方法：使用外泌体提取试剂盒提取原代胶质母细胞瘤来源外泌体,通过透射电子显微镜、纳米粒度电位仪和Western blotting对外泌体进行鉴定;采用Western blotting检测外泌体中HMGB1的表达量;通过qRT-PCR、Western blotting、克隆球计数检测外泌体对胶质瘤干细胞形成的影响;siRNA敲低HMGB1的表达水平,并通过qRT-PCR、Western blotting、克隆球计数检测外泌体中HMGB1对胶质瘤干细胞形成的影响。结果：原代胶质瘤细胞可以分泌外泌体到肿瘤微环境并且外泌体中存在HMGB1;原代胶质瘤细胞来源外泌体可以上调邻近胶质瘤细胞干性相关分子CD133、OCT4、NANOG、SOX2的表达并促进干细胞克隆球的形成;通过siRNA敲低原代胶质瘤细胞HMGB1的表达后,外泌体中HMGB1的含量降低并且外泌体促进胶质瘤干细胞形成的作用减弱。结论：胶质瘤细胞来源外泌体可以通过HMGB1促进胶质瘤干细胞的形成。     

脂肪干细胞外泌体对子宫内膜癌HEC-251细胞增殖和凋亡的影响

为了探讨脂肪干细胞外泌体对子宫内膜癌HEC-251细胞增殖和凋亡的影响及分子机制,本研究从人体脂肪组织中分离脂肪干细胞并提取纯化其外泌体。实验分为3组:对照组、脂肪干细胞外泌体组和TGF-β阻断剂组。CCK8检测脂肪干细胞外泌体及TGF-β阻断剂对子宫内膜癌HEC-251细胞活力;流式检测脂肪干细胞外泌体及TGF-β阻断剂对子宫内膜癌细胞凋亡的影响;Western blotting检测脂肪干细胞外泌体及TGF-β阻断剂对子宫内膜癌细胞Smad2、p-smad2、Bcl2和TGF-β蛋白表达水平。CCK8结果显示,脂肪干细胞外泌体能够显著增强子宫内膜癌HEC-251细胞的增殖能力,TGF-β阻断剂能够显著抑制外泌体对子宫内膜癌的增殖促进作用,流式检测结果显示脂肪干细胞外泌体能够显著抑制子宫内膜癌HEC-251细胞的凋亡,TGF-β阻断剂能够显著抑制外泌体对子宫内膜癌的细胞凋亡的抑制作用,Western blotting检测显示脂肪干细胞外泌体能够显著抑制子宫内膜癌HEC-251细胞p-smad2、Bcl2和TGF-β蛋白表达。初步研究表明,脂肪干细胞外泌体通过促进TGF-β/smad通路促进子宫内膜癌HEC-251细胞增殖,抑制细胞凋亡。     

膀胱癌细胞来源外泌体的提取与鉴定

目的:提取并鉴定膀胱癌5637细胞来源外泌体。方法:收集膀胱癌5637细胞培养上清液,采用多步骤离心法提取膀胱癌5637细胞外泌体。透射电子显微镜观察外泌体形态及颗粒直径。Bradford法定量外泌体蛋白含量。蛋白质免疫印迹鉴定外泌体标志蛋白。结果:20 m L 5637细胞培养基可收集约50-80μg外泌体。膀胱癌5637细胞来源外泌体呈典型的茶杯样形态,外泌体颗粒直径大约在30-150 nm。膀胱癌5637细胞外泌体提取物中可检测到标志蛋白CD63、TSG101、Hsp70和Hsp90表达。结论:多步骤离心法可以用于提取膀胱癌5637细胞外泌体,为后续开展膀胱癌5637细胞外泌体作用与机制的研究奠定了基础。     

丹皮酚对PM2.5诱导的支气管上皮细胞IL-8表达的影响

目的:观察丹皮酚对PM2.5诱导支气管上皮细胞(BEAS-2B)分泌IL-8的影响。方法:BEAS-2B细胞传代培养,分别加入丹皮酚(15μmol/L、30μmol/L)预处理1 h,ELISA法检测不同浓度的PM2.5悬液(25μg/m L、50μg/m L、100μg/m L)对BEAS-2B细胞分泌IL-8蛋白的影响。结果:随着PM2.5悬液浓度的升高,BEAS-2B细胞上清液中IL-8水平逐渐增高;丹皮酚干预后能够显著降低BEAS-2B细胞上清液中IL-8水平(P0.05),并呈浓度依赖性。结论:丹皮酚具有抑制PM2.5诱导的BEAS-2B细胞分泌IL-8蛋白表达的作用,并呈浓度依赖性。     

采用TOPOTA克隆技术构建人CC10质粒并在BEAS-2B细胞中的表达

目的:采用TOPO TA克隆技术构建及鉴定人CC10基因表达载体,并在支气管上皮细胞系BEAS-2B细胞中获得稳定表达,初步探讨CC10在呼吸道上皮细胞炎症反应中的作用。方法:提取人下鼻甲组织的总RNA,逆转录反应生成c DNA,再用PCR方法扩增出含人CC10编码区全长的DNA片段,将PCR产物直接连接到pc DNA3.1/V5-His TOPO TA载体中,转化至大肠杆菌后经筛选鉴定出CC10表达载体,用脂质体法将CC10质粒转染到BEAS-2B,细胞免疫荧光法检测CC10蛋白的表达。随后用促炎细胞因子IL-1β刺激转染空质粒和CC10质粒的BEAS-2B细胞,用实时定量RT-PCR和ELISA检测炎性趋化因子RANTES m RNA和蛋白的表达。结果:目的基因与TOPO TA载体在室温下5 min的连接反应效率91.7%,用酶切法鉴定质粒并测序,人CC10基因成功克隆到真核细胞表达载体pc DNA3.1中,CC10蛋白在BEAS-2B细胞中无表达,但在体外转染CC10质粒后CC10蛋白表达明显增高。转染CC10质粒的BEAS-2B细胞可抑制IL-1β诱导的RANTES m RNA和蛋白的表达。结论:利用TOPO TA克隆技术可高效、快速的构建人CC10基因表达载体,并能够在BEAS-2B细胞中获得稳定表达,CC10蛋白在呼吸道上皮细胞中可发挥抗炎作用。     

网状内皮增生症病毒感染改变外泌体蛋白质组成和免疫调节功能

采用间接免疫荧光、透射电镜观察、Western blot、质谱分析、酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)和流式细胞术分析技术检测了网状内皮增生症病毒(reticuloendotheliosis virus,REV)感染DF-1细胞的情况、感染细胞分泌外泌体(exosome)的形态大小、标志蛋白质水平以及REV对外泌体蛋白质组成和免疫功能的影响。结果显示,接毒成功的DF-1细胞胞质中有明显的绿色荧光出现。提取的外泌体呈杯状,平均直径大小在50~100 nm,表达标志蛋白质Hsp70和Tsg101。与正常未感染细胞相比,REV感染细胞分泌的外泌体共存在58种表达水平差异的蛋白质。其中上调的46种,包括REV的env蛋白gp90和gag-pol在内的4种蛋白质,下降的12种。这些外泌体同时具有明显的促进脾细胞分泌IL-10的作用;对CD4+T和CD8+T细胞均具有明显的抑制性。REV感染改变了外泌体的蛋白质组成,尤其是携带了REV的重要成分,该成分与引起的外泌体抑制性免疫调节作用密切相关。     

过表达IDO的骨髓间充质干细胞分泌外泌体下调树突状细胞免疫促进分子表达和上调Treg细胞数量

目的探讨过表达吲哚胺2, 3-双加氧酶(indoleamine 2, 3-dioxy genase, IDO)大鼠骨髓间充质干细胞(bone marrow mesenchy mal stem cells, BMSCs))分泌外泌体(exosome, ES)的免疫抑制调节作用。方法构建过表达IDO大鼠骨髓间充质干细胞提取分泌的外泌体做为实验组,将空载体大鼠骨髓间充质干细胞、大鼠骨髓间充质干细胞分泌的外泌体做为对照组,将实验组及对照组分泌的外泌体分别与树突状细胞(DC细胞)、T细胞体外共培养48h后采用流式细胞检测DC细胞表面免疫调节分子表达及T细胞亚群分子标志物表达,同时采用RT-PCR检测DC细胞中IDO表达量。结果过表达IDO的BMSCs分泌的外泌体与DC细胞共培养使DC细胞CD40、CD86、CD80、MHCII、CD45RA+CD45RB、OX62等免疫促进分子表达率降低,而CD274表达率升高,同时DC细胞中IDO表达量增多;而过表达IDO的BMSCs分泌的外泌体与T细胞共培养组使T细胞亚群中的Treg细胞数量增加,CD4阳性T细胞变化无规律性,但CD8阳性T细胞减少。结论过表达IDO-BMSCs分泌的外泌体可通过抑制DC活性和上调Treg细胞数量,产生免疫抑制作用。     

Montelukast suppresses epithelial to mesenchymal transition of bronchial epithelial cells induced by eosinophils

Epithelial to mesenchymal transition (EMT) is a mechanism by which eosinophils can induce airway remodeling. Montelukast, an antagonist of the cysteinyl leukotriene receptor, can suppress airway remodeling in asthma. The purpose of this study was to evaluate whether montelukast can ameliorate airway remodeling by blocking EMT induced by eosinophils. EMT induced was assessed using a co-culture system of human bronchial epithelial cells and human eosinophils or the eosinophilic leukemia cell lines, Eol-1. Montelukast inhibited co-culture associated morphological changes of BEAS-2b cells, decreased the expression of vimentin and collagen I, and increased the expression of E-cadherin. Montelukast mitigated the rise of TGF-β1 production and Smad3 phosphorylation. Co-culture of human eosinophils with BEAS-2B cells significantly enhanced the production of CysLTs compared with BEAS-2B cells or eosinophils alone. The increase of CysLTs was abolished by montelukast pre-treatment. Montelukast had similar effects when co-culture system of Eol-1 and BEAS-2B was used. This study showed that montelukast suppresses eosinophils-induced EMT of airway epithelial cells. This finding may explain the mechanism of montelukast-mediated amelioration of airway remodeling in bronchial asthma.     

Rhinovirus-induced MMP-9 expression is dependent on Fra-1, which is modulated by formoterol and dexamethasone

Matrix metalloproteinase-9 is implicated in airway inflammation and airway remodeling in asthma. We have previously confirmed that human rhinovirus-16 (HRV-16) infection increases MMP-9 expression both in vivo and in vitro. However, the role of the AP-1 sites within the MMP-9 promoter and the effect of commonly used asthma pharmacotherapies in modulating human rhinovirus (HRV)-induced MMP-9 production have not yet been elucidated. Experiments were performed in vitro in the human bronchial epithelial (HBE) cell line BEAS-2B and in primary HBE cells obtained from non-transplanted lungs. Using site-directed mutagenesis approaches, AP-1 sites were found to be necessary for HRV-induced MMP-9 promoter drive. EMSAs and supershift assays identified complexes consisting of Fos-related Ag-1 (Fra-1) in addition to other AP-1 subunits. Small interfering RNA approaches indicated that Fra-1 was induced upon HRV-16 infection in BEAS-2B cells and was necessary for MMP-9 expression in both BEAS-2B and primary HBE cells. Inhibition of MEK1/2 activity using PD98059 and U0126 reduced Fra-1 expression, DNA binding, MMP-9 promoter drive, and MMP-9 protein production. The long-acting β(2)-agonist formoterol and the glucocorticoid dexamethasone significantly reduced HRV-induced ERK phosphorylation, Fra-1, and MMP-9 expression in BEAS-2B cells. These data indicate that HRV-induced activation of the MEK/ERK MAPK pathway and Fra-1 expression are necessary for the upregulation of MMP-9 and can be modulated by two distinct but commonly used asthma pharmacotherapies. Together, these results offer insights into the mechanisms by which long-acting β(2)-agonists and glucocorticoids might reduce HRV-related asthma exacerbations.     

前列腺癌细胞来源的外泌体诱导巨噬细胞极化

外泌体(exosome)是直径约30～150 nm的由细胞分泌的一种具有生物学活性的囊泡。有些来自癌细胞的外泌体可以将巨噬细胞(macrophages,Mφ)极化为M2亚型,但前列腺癌细胞来源的外泌体在巨噬细胞极化中的作用仍缺乏研究。本研究采用超滤法提取前列腺癌细胞PC-3M-2B4和PC-3M-IE8条件培养基中的外泌体(PCa-exo)。分别用透射电子显微镜、纳米粒径分析及Western印迹对外泌体形态、颗粒大小和表面的特异性分子标志进行分析鉴定。用PKH67标记外泌体,观察PCa-exo能否被巨噬细胞吸收。免疫荧光分析PCa-exo处理巨噬细胞后,M2型巨噬细胞表面分子标志CD206的表达差异。用q-PCR观察PCa-exo诱导后的巨噬细胞中IL-10、IL-1β等细胞因子的表达。电镜、Western印迹和纳米粒径分析的结果显示,PCa-exo形态多为圆形,直径约为40～150 nm,PCa-exo能被巨噬细胞大量吸收。PCa-exo诱导后,巨噬细胞中CD206荧光表达显著增高,IL-10、IL-1β及IL-12等炎症因子的表达水平与M2/TAM亚型巨噬细胞的表达谱一致。本研究表明,前列腺癌细胞来源的外泌体能诱导巨噬细胞极化为M2表型。     

Isoproterenol suppresses cytokine-induced RANTES secretion in human lung epithelial cells through the inhibition of c-jun N-terminal kinase pathway

It has been reported that beta2-agonists may potentially exert some anti-inflammatory action in addition to bronchodilation that may contribute to their beneficial effects on asthma control. Bronchial epithelial cells are well known to respond to a range of stimuli by producing various biologically active mediators that can influence airway inflammation. RANTES (regulated on activation, normal T cells expressed and secreted) plays an important role in the pathophysiology of airway inflammation of asthmatics through its chemotactic activity for eosinophils. In this study, the authors investigated whether cytokine-induced RANTES release from BEAS-2B human bronchial epithelial cells could be modulated by beta-agonist isoproterenol (ISO). The possible involvement of c-jun N-terminal kinase (JNK) pathway was also studied. Combination of tumor necrosis factor-alpha and interleukin-1beta (cytokine mix) increased RANTES release from BEAS-2B cells and stimulated JNK activity. Similar to JNK inhibitor SP600125, ISO inhibited not only the production of RANTES but also the activation of JNK pathway in cytokine mix-stimulated BEAS-2B cells. The effect of ISO was mediated by the beta2-adrenoceptor, since it was blocked by ICI 118,551, a selective beta2-receptor antagonist, but not by atenolol, a selective beta1-receptor antagonist. Adenylyl cyclase activator forskolin reproduced the effects of ISO. Isoproterenol was found to inhibit the release of RANTES from the human bronchial epithelial cells, at least in part, through the inhibition of JNK signaling pathway.     

LTD₄ induces HB-EGF-dependent CXCL8 release through EGFR activation in human bronchial epithelial cells

Airway epithelial cells release proinflammatory mediators that may contribute to airway remodeling and leukocyte recruitment. We explored the hypothesis that leukotriene D? (LTD?) may trigger the release of proremodeling factors through activation of the EGF receptor (EGFR). We particularly focused on the effects of LTD? on release of heparin-binding EGF-like factor (HB-EGF) and IL-8 (CXCL8), a potent neutrophil chemoattractant that may be released downstream of EGFR activation. To address this hypothesis, both primary (NHBE) and transformed bronchial human epithelial cells (BEAS-2B) were grown on an air-liquid interface and stimulated with LTD?. HB-EGF and CXCL8 were evaluated by ELISA in cell culture supernatants. To explore the EGFR signaling pathway, we used a broad-spectrum matrix metalloproteinase (MMP) inhibitor, GM-6001, two selective EGFR tyrosine kinase inhibitors, AG-1478 and PD-153035, an HB-EGF neutralizing antibody, and a specific small interfering RNA (siRNA) against the EGFR. Expression of the CysLT? cysteinyl leukotriene receptor was demonstrated by RT-PCR and immunocytochemistry in both BEAS-2B and NHBE cells. Four hours after stimulation with LTD?, HB-EGF and CXCL8 were significantly increased in cell culture supernatant. GM-6001 and montelukast, a specific CysLT? receptor antagonist, blocked the LTD?-induced increase in HB-EGF. All inhibitors/antagonists decreased LTD?-induced CXCL8 release. siRNA against EGFR abrogated CXCL8 release following stimulation with LTD? and exogenous HB-EGF. These findings suggest LTD? induced EGFR transactivation through the release of HB-EGF in human bronchial epithelial cells with downstream release of CXCL8. These effects may contribute to epithelial-mediated airway remodeling in asthma and other conditions associated with cysteinyl leukotriene release.     

Exosomes derived from RPE cells under oxidative stress mediate inflammation and apoptosis of normal RPE cells through Apaf1/caspase-9 axis

This study aims to explore the effects of exosomes, secreted by retinal pigment epithelial (RPE) cells under oxidative stress (OS), on apoptosis and inflammation of normal RPE cells. Exosomes secreted by normal RPE cells (named as exo) and rotenone (2.5 µmol/L) stimulated RPE cells (named as rot-exo) were isolated and extracted by multi-step differential centrifugation for morphology observation under a transmission electron microscopy. pcDNA3.1a, pcDNA3.1a-Apaf1, and p3xFlag-CMV-caspase-9 plasmids were constructed and transfected into ARPE-19 cells. Exosomes secreted by ARPE-19 cells were injected into the vitreous body of rats to verify the effect of Apaf1 and caspase-9 on cell apoptosis and inflammation. Co-immunoprecipitation was applied to clarify the interaction of Apaf1 with caspase-9. Exosomes secreted by rotenone stimulated ARPE-19 cells could induce cell apoptosis, oxidative injury, and inflammation in ARPE-19 cells. Exosomes secreted under OS can damage retinal functions of rats and have upregulated expression of Apaf1. Overexpression of Apaf1 in exosomes secreted under OS can cause the inhibition of cell proliferation, the increase of cell apoptosis and elicitation of inflammatory response in ARPE-19 cells. Exosomes derived from ARPE-19 cells under OS regulate Apaf1 expression to increase cell apoptosis and to induce oxidative injury and inflammatory response through a caspase-9 apoptotic pathway.