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。     

大熊猫脐带间充质干细胞外泌体的分离鉴定及miRNAs富集分析

大熊猫脐带间充质干细胞(Umbilical cord mesenchymal stem cells, UC-MSCs)通过旁分泌所释放的外泌体在大熊猫保健与疾病治疗方面具有一定的应用前景。本研究旨在建立大熊猫UC-MSCs外泌体分离方法,开展生物学特征分析和分子鉴定,并研究UC-MSCs外泌体中miRNAs的种类与功能。采用超速离心法从大熊猫UCMSCs培养上清中成功分离外泌体,通过透射电子显微镜进行形态学观察,纳米颗粒跟踪分析仪检测粒径大小,蛋白免疫印迹法检测特异性分子标记表达。采用Small RNA测序技术对UC-MSCs外泌体中的miRNAs进行鉴定,并对其靶基因进行了预测与功能分析。结果显示,大熊猫UC-MSCs外泌体呈圆形杯托状结构,直径为(79.15±4.81) nm,外泌体标志蛋白CD81与TSG101呈阳性表达而CALNEXIN呈阴性表达。大熊猫UC-MSCs外泌体中的miRNA主要为miR-148-3p (30.28%)与miR-21-5p (21.72%)。本研究首次从大熊猫UC-MSCs培养上清中分离出外泌体,并对其所含的miRNAs进行富集分析及功能预测,为大熊...     

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。     

肝缺血再灌注大鼠血清外泌体引起海马和大脑皮质损伤

本文旨在探究大鼠肝缺血再灌注(ischemia/reperfusion, I/R)后血清中外泌体的变化及其在海马和大脑皮质损伤中的作用。采用随机数字表法将清洁级雄性Sprague-Dawley (SD)大鼠分为4组:假手术组(S组)、肝缺血再灌注组(I/R组)、S组大鼠血清外泌体处理组(ES组)和I/R组大鼠血清外泌体处理组(EI组)。ES组和EI组为正常大鼠经尾静脉分别注射S组和I/R组大鼠血清中的外泌体各100μL。另取3只正常大鼠经尾静脉注射PKH26红色荧光标记的外泌体,利用免疫荧光显微镜观察大鼠脑组织中荧光表达情况。应用透射电子显微镜观察外泌体的形态和大小;免疫蛋白印迹法检测外泌体标记蛋白CD63和CD9的表达情况;通过血清学和组织学指标的检测,观察肝及脑组织损伤情况及海马和大脑皮质组织细胞凋亡和氧化应激水平。结果显示:外泌体是直径为30～100 nm的圆形或椭圆形膜性囊泡。与S组比较,I/R组大鼠血清中外泌体含量增加(P 0.05)。外泌体具有血脑屏障通透性,可自外周经血液循环进入脑组织。I/R组血清肝功能指标较S组明显升高(P 0.05)。与S组比较,ES组脑组织损伤程度及海马和大脑皮质组织细胞凋亡和氧化应激水平无显著变化;与S组、ES组比较,I/R组、EI组血清脑损伤标志物水平、海马和大脑皮质组织中凋亡指数、氧化应激水平升高,但EI组以上指标均低于I/R组(P 0.05)。因此,大鼠肝I/R可导致海马和大脑皮质组织损伤,而且其介导的血清外泌体含量增加在其中发挥了重要作用。     

结直肠癌细胞外泌体激活肝星状细胞为癌相关成纤维细胞

目的:探讨结直肠癌(CRC)细胞上清外泌体对肝星状细胞(HSC)细胞的影响。方法:通过超高速离心结合过滤法提取和纯化CRC细胞上清外泌体,然后以透射电电子显微镜(TEM)、纳米颗粒跟踪分析仪(NTA)和蛋白免疫印迹(WB)实验鉴定所提取的外泌体的形态、大小、粒径分布,以及外泌体表面标志蛋白HSP90和TSG101。再通过激光共聚焦显微镜(LSCM)观察荧光标记的外泌体被HSC细胞摄取的情况。以WB实验验证CRC细胞上清外泌体处理后的HSC中成纤维细胞活化蛋白(FAP)和α-平滑肌肌动蛋白(α-SMA)的表达水平。结果:TEM显示结直肠癌细胞外泌体呈"茶托样"杯型或类圆形囊泡样结构;NTA分析发现结直肠癌细胞外泌体直径峰值和大小分布范围分别为57 nm和30-150 nm;WB显示外泌体表面标志蛋白HSP90和TSG101均为阳性。LSCM观察发现Di O标记的外泌体(绿色),能够被Dil标记的HSC(红色)摄取。CRC细胞上清外泌体处理后的HSC中FAP和α-SMA表达水平较对照组显著升高。结论:HSC与CRC细胞上清外泌体共孵育后能够被激活成癌相关成纤维细胞。     

干细胞外泌体对内皮细胞增殖迁移和血管生成的影响

[目的]探究脂肪间充质干细胞来源的外泌体对人脐静脉内皮细胞的迁移和血管生成的影响。[方法]利用超速离心法提取脂肪间充质干细胞来源的外泌体;通过透射电子显微镜观察外泌体的形貌;通过免疫印迹法检测外泌体的标志蛋白;通过动态光散射法检测外泌体水合粒径;通过CCK-8法检测外泌体对HUVECs增殖的作用;通过划痕实验和Transwell实验检测外泌体对HUVECs迁移的影响;通过血管生成实验评估外泌体诱导HUVECs生成血管的能力。[结果]脂肪间充质干细胞外泌体的平均水合粒径约为151.9±12.3 nm,含有外泌体的标志蛋白CD63、TSG101。外泌体浓度为30μg/mL时,共孵育48 h后,EXO组HUVECs的增殖率高于NC组14%;划痕实验中NC组的平均迁移率约为0.45±0.05,EXO组约为0.63±0.05,EXO组的迁移率显著高于NC组约为0.18±0.07,而Transwel中48 h时NC组的单位面积平均转移细胞数约为167±24,EXO组约为728±49。4 h的成血管实验显示外泌体组管的NC组平均结点数约为495±52,EXO组约为658±76;NC组单位图像平均分支...     

肝细胞癌患者血清外泌体microRNA表达鉴定

目的:探讨肝癌细胞外泌体中差异表达的microRNAs(miRNAs)在肝细胞癌(HCC)诊断中的应用价值。方法:通过高通量测序筛选肝癌细胞外泌体中差异表达的miRNAs。实时定量PCR验证差异表达分子;检测差异表达的miRNAs在健康人(Health)、慢性乙型肝炎患者(CHB)、肝硬化患者(LC)及乙型肝炎病毒阳性的肝细胞癌患者(HCC)血清外泌体中的表达。结果:高通量测序筛选到肝癌细胞外泌体中差异表达的miRNA共88种,其中58种表达上调,30种表达下调。选择其中8种差异表达的miRNAs进行q RT-PCR验证,结果显示,此8种miRNAs在细胞上清外泌体、细胞内、癌与癌旁组织中的表达趋势与测序结果一致。miR-221-3p和miR-224-5p在HCC组外泌体中的表达水平显著高于Health组、CHB组和LC组(P0.01),miR-124-3p和let-7a-5p在HCC组外泌体中的表达水平显著低于其他各组(P0.05)。四个组中,miR-21-5p、miR-191-5p、miR-34a-5p和miR-122-5p的表达水平不存在显著性差异(P0.05)。结论:血清外泌体中的miR-221-3p、miR-224-5p、miR-124-3p和let-7a-5p可能成为肝细胞癌的候选标志物。     

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

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。     

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。     

聚乙二醇沉淀剂有效分离尿外泌体

尿外泌体是病毒大小的胞外囊泡,是非侵入性获得肾及泌尿生殖道细胞生理病理信息的重要靶标。聚乙二醇沉淀 剂可经济高效地分离富集血清等外泌体,但未见用于尿外泌体富集的详细报道。本研究采用聚乙二醇沉淀剂分离鉴定尿外泌体,并对其RNA组分进行检测,以期建立一个经济、高效、简便的尿外泌体分离富集方法。采集10例健康志愿者晨尿20 mL,聚乙二醇沉淀剂分离尿外泌体。透射电镜观察到直径30～100 nm双层膜包绕的囊性小泡,中央有直径5～15 nm高电子密度区。Western印迹检测到外泌体标记蛋白CD63、CD9、TSG101、ADAM10和内标蛋白β-肌动蛋白的表达。纳米粒径仪测定粒子直径介于30～130 nm,并可见25.37 nm和95.07 nm二个粒子峰。qRT-PCR扩增得到β-肌动蛋白和RNU6 RNA产物带。上述结果表明,聚乙二醇沉淀剂可分离富集尿外泌体,该法简单、高效,不需要超速离心机等昂贵设备,且采用该法富集到的外泌体可用于后续蛋白质与核酸分析。该方法可望加速液体活检应用,尤其是肾及泌尿生殖道病变的无创检测。     

microRNA modified tumor-derived exosomes as novel tools for maturation of dendritic cells

Tumor-derived exosomes (TEX) are known by their immune suppression effects as well as initiation mediators in cancer progression and metastasis. Meanwhile, they are appropriate sources to induce immunity against tumor cells, as consist of tumor specific and associated antigens. The aim of the current study is modifying TEX with microRNA miR-155, miR-142, and let-7i, to enhance their immune stimulation ability and induce potent dendritic cells (DC). For this, exosomes were isolated from mouse mammalian breast cancer cell line; 4T1, and subjected to miR-155, miR-142, and let-7i by electroporation. Immature DCs were generated from mouse bone marrow in the presence of interleukin-4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF). To mature DCs, lipopolysaccharide (LPS), TEX, and modified TEX were used. The expression level of miRNAs and their target genes (IL-6, IL-17, IL-1b, TGFβ, SOCS1, KLRK1, IFNγ, and TLR4) was determined. TEX were nanovesicles with spheroid morphology which expressed CD81, CD63, and TSG101, as exosome markers, at protein level. MHCII, CD80, and CD40 as maturation markers were assessed by flow cytometry. Overexpression of miRNAs were confirmed in exosomes and mDCs. Up and downregulation of target genes confirmed the gene network in DC maturation. We found that Let-7i could efficiently induce the DC maturation, as well as miR-142 and miR-155 have enhancing effects. These findings reveal that the modified TEX would be a hopeful cell-free vaccine for the cancer treatment.     

MiR-122在AldoA介导的ob/ob小鼠肝脏代谢中的作用研究

本实验室前期研究发现,2型糖尿病动物模型ob/ob小鼠血清中miR-122的含量较正常C57BL/6小鼠显著升高.本文进一步研究肝脏特异性miR-122及其靶蛋白AldoA(果糖1,6-二磷酸醛缩酶A)在ob/ob小鼠肝脏代谢中的作用.首先,经qRT-PCR技术检测发现ob/ob小鼠肝脏miR-122水平较C57BL/6小鼠显著下降,而Western blotting分析发现ob/ob小鼠肝脏其靶蛋白AldoA的表达水平显著上升.进一步以miR-122分子转染293T细胞后收集其分泌的微囊泡(microvesicles,MVs),经qRT-PCR检测确认后采用特异性荧光染料DiI-C18标记MVs,以不同剂量尾静脉注射BALB/c小鼠体内,不同时间点取肝组织做冰冻切片.在荧光显微镜下观察证实,包裹有miR-122的MVs通过循环系统进入肝脏,同时qRT-PCR定量分析发现肝组织中miR-122含量显著升高,而蛋白质印迹检测发现其靶蛋白AldoA在肝脏中表达显著下降.AldoA主要催化糖酵解途径中果糖1,6-二磷酸和磷酸二羟丙酮及甘油醛-3-磷酸之间的转变,miR-122靶向作用AldoA可能在2型糖尿病的发生发展中发挥重要作用.     

Differential expression of circular RNAs in bone marrow-derived exosomes from essential thrombocythemia patients

Circular RNAs (circRNA) are closely associated with the pathogenesis of various hematological diseases. However, little is known about the potential functions of circRNAs in essential thrombocythemia (ET) development. The circRNA profile alterations in the bone marrow of ET patients were mainly investigated in this study. The sizes of exosomes derived from human bone marrow tissues were validated by the nanoparticle tracking analysis (NTA) method. CD63 and TSG101 expressions in exosomes were analyzed by western blot analysis. The profiles and differential expression of circRNAs in bone-derived exosomes were characterized by high-throughput sequencing. Herein, circular structures and expression of circRNAs were verified by Sanger sequencing and real-time polymerase chain reaction, respectively. The circRNA-miRNA-mRNA networks were predicted using the Cytoscape software. And we detected the effect of circ_0014614 on the transformation of K562 cells into megakaryocytes. Exosomes derived from the bone marrow of ET patients and healthy volunteers showed a diameter between 70 and 140 nm and expressed high CD63 and TSG101. Meanwhile, the circRNA profiles were significantly altered in bone marrow–derived exosomes from ET patients, among which circDAP3, circASXL1, and circRUNX1 were significantly downregulated in ET patients, thus conferring a new insight into the role of circRNAs in the pathogenesis of ET. Besides this, circRNA-encoding genes and miRNA-mRNA networks targeted by this three circRNA were involved in various biological processes and signaling pathways. And circ_0014614 could inhibit K562 cells' differentiation into megakaryocytes. The predictions of the potential function of these three differentially expressed circRNAs along with their interaction with specific miRNAs could provide a basis for circRNA-based ET diagnosis and treatment.     

一种快速提取组织外泌体的分离富集方法

本文提供一种快速提取组织外泌体的分离富集方法。通过将目标组织用机械法切碎,加入组织消化酶进行组织解离和过滤,将获得的组织细胞悬液依次进行差速离心、超离、尺寸排阻和超滤,实现组织高质量外泌体的富集纯化。组织解离方法比较试验中,采用组织消化酶解离组织得到的蛋白质含量更高,获得的外泌体组织来源的蛋白质污染小。富集小鼠心组织、小鼠肝组织、小鼠肾组织、人结肠癌组织、人乳腺癌组织和动脉粥样硬化组织的外泌体,并对其进行纳米粒径追踪和透射电镜观察。结果显示,外泌体的粒径均在30~150 nm内,结构清晰明确。对小鼠肝组织富集的外泌体进行蛋白质印迹分析。结果显示,阳性蛋白质标志物CD9、ALIX和CD63的表达,TSG101弱表达,阴性蛋白质标志物Calnexin无表达。本方法集合多种分离措施,能够达到分离纯化外泌体的作用,同时简化了分离组织外泌体的步骤,相对于其他方法,全程只需要4~5 h,节省了富集时间,所富集的外泌体纯度高、可溶性杂蛋白质污染小,实用性更加广泛。使用微量组织样本富集的外泌体即可满足后续纳米粒径追踪、蛋白质印迹、透射电镜和转录物组等分析。     

Comparative proteomics evaluation of plasma exosome isolation techniques and assessment of the stability of exosomes in normal human blood plasma

Exosomes are nanovesicles released by a variety of cells and are detected in body fluids including blood. Recent studies have highlighted the critical application of exosomes as personalized targeted drug delivery vehicles and as reservoirs of disease biomarkers. While these research applications have created significant interest and can be translated into practice, the stability of exosomes needs to be assessed and exosome isolation protocols from blood plasma need to be optimized. To optimize methods to isolate exosomes from blood plasma, we performed a comparative evaluation of three exosome isolation techniques (differential centrifugation coupled with ultracentrifugation, epithelial cell adhesion molecule immunoaffinity pull‐down, and OptiPrep^TM density gradient separation) using normal human plasma. Based on MS, Western blotting and microscopy results, we found that the OptiPrep^TM density gradient method was superior in isolating pure exosomal populations, devoid of highly abundant plasma proteins. In addition, we assessed the stability of exosomes in plasma over 90 days under various storage conditions. Western blotting analysis using the exosomal marker, TSG101, revealed that exosomes are stable for 90 days. Interestingly, in the context of cellular uptake, the isolated exosomes were able to fuse with target cells revealing that they were indeed biologically active.     

Exosomes Are Unlikely Involved in Intercellular Nef Transfer

HIV-1 Nef is an important pathogenic factor for HIV/AIDS pathogenesis. Several recent studies including ours have demonstrated that Nef can be transferred to neighboring cells and alters the function of these cells. However, how the intercellular Nef transfer occurs is in dispute. In the current study, we attempted to address this important issue using several complementary strategies, a panel of exosomal markers, and human CD4+ T lymphocyte cell line Jurkat and a commonly used cell line 293T. First, we showed that Nef was transferred from Nef-expressing or HIV-infected Jurkat to naïve Jurkat and other non-Jurkat cells and that the transfer required the membrane targeting function of Nef and was cell density-dependent. Then, we showed that Nef transfer was cell-cell contact-dependent, as exposure to culture supernatants or exosomes from HIV-infected Jurkat or Nef-expressing Jurkat and 293T led to little Nef detection in the target cells Jurkat. Thirdly, we demonstrated that Nef was only detected to be associated with HIV virions but not with acetylcholinesterase (AChE+) exosomes from HIV-infected Jurkat and not in the exosomes from Nef-expressing Jurkat. In comparison, when it was over-expressed in 293T, Nef was detected in detergent-insoluble AChE+/CD81^low/TSG101^low exosomes, but not in detergent-soluble AChE-/CD81^high/TSG101^high exosomes. Lastly, microscopic imaging showed no significant Nef detection in exosomal vesicle-like structures in and out 293T. Taken together, these results show that exosomes are unlikely involved in intercellular Nef transfer. In addition, this study reveals existence of two types of exosomes: AChE+/CD81^low/TSG101^low exosomes and AChE-/CD81^high/TSG101^high exosomes.