外泌体传递的非编码RNA在鼻咽癌发生发展中的作用

外泌体作为细胞间信息交流的重要介质,通过传递多种生物信号分子在人类癌症的发生发展中起着关键的调控作用。非编码RNA参与调控多种肿瘤恶性生物学行为,同时可反映肿瘤的进展及用于疗效评估。鼻咽癌是中国南方高发的EB病毒感染相关头颈部恶性肿瘤,本文对外泌体来源的非编码RNA在鼻咽癌发生发展中所扮演的角色及其潜在的临床应用价值进行综述。     

Pivoting Novel Exosome-Based Technologies for the Detection of SARS-CoV-2

The National Institutes of Health (NIH) launched the Rapid Acceleration of Diagnostics (RADx) initiative to meet the needs for COVID-19 diagnostic and surveillance testing, and to speed its innovation in the development, commercialization, and implementation of new technologies and approaches. The RADx Radical (RADx-Rad) initiative is one component of the NIH RADx program which focuses on the development of new or non-traditional applications of existing approaches, to enhance their usability, accessibility, and/or accuracy for the detection of SARS-CoV-2. Exosomes are a subpopulation of extracellular vesicles (EVs) 30–140 nm in size, that are critical in cell-to-cell communication. The SARS-CoV-2 virus has similar physical and molecular properties as exosomes. Therefore, the novel tools and technologies that are currently in development for the isolation and detection of exosomes, may prove to be invaluable in screening for SARS-CoV-2 viral infection. Here, we describe how novel exosome-based technologies are being pivoted for the detection of SARS-CoV-2 and/or the diagnosis of COVID-19. Considerations for these technologies as they move toward clinical validation and commercially viable diagnostics is discussed along with their future potential. Ultimately, the technologies in development under the NIH RADx-Rad exosome-based non-traditional technologies toward multi-parametric and integrated approaches for SARS-CoV-2 program represent a significant advancement in diagnostic technology, and, due to a broad focus on the biophysical and biochemical properties of nanoparticles, the technologies have the potential to be further pivoted as tools for future infectious agents.     

Exosomes Derived from Glioma Cells under Hypoxia Promote Angiogenesis through Up-regulated Exosomal Connexin 43

Glioblastoma multiform (GBM) is a highly aggressive primary brain tumor. Exosomes derived from glioma cells under a hypoxic microenvironment play an important role in tumor biology including metastasis, angiogenesis and chemoresistance. However, the underlying mechanisms remain to be elucidated. In this study, we aimed to explore the role of connexin 43 on exosomal uptake and angiogenesis in glioma under hypoxia. U251 cells were exposed to 3% oxygen to achieve hypoxia, and the expression levels of HIF-1α and Cx43, involved in the colony formation and proliferation of cells were assessed. Exosomes were isolated by differential velocity centrifugation from U251 cells under normoxia and hypoxia (Nor-Exos and Hypo-Exos), respectively. Immunofluorescence staining, along with assays for CCK-8, tube formation and wound healing along with a transwell assay were conducted to profile exosomal uptake, proliferation, tube formation, migration and invasion of HUVECs, respectively. Our results revealed that Hypoxia significantly up-regulated the expression of HIF-1α in U251 cells as well as promoting proliferation and colony number. Hypoxia also increased the level of Cx43 in U251 cells and in the exosomes secreted. The uptake of Dio-stained Hypo-Exos by HUVECs was greater than that of Nor-Exos, and inhibition of Cx43 by ^37,43gap27 or lenti-Cx43-shRNA efficiently prevented the uptake of Hypo-Exos by recipient endothelial cells. In addition, the proliferation and total loops of HUVECs were remarkably increased at 24 h, 48 h, and 10 h after Hypo-Exos, respectively. Notably, ^37,43gap27, a specific Cx-mimetic peptide blocker of Cx37 and Cx43, efficiently alleviated Hypo-Exos-induced proliferation and tube formation by HUVECs. Finally, ^37,43gap27 also significantly attenuated Hypo-Exos-induced migration and invasion of HUVECs. These findings demonstrate that exosomal Cx43 contributes to glioma angiogenesis mediated by Hypo-Exos, and suggests that exosomal Cx43 might serve as a potential therapeutic target for glioblastoma.     

miR-106调控CC趋化因子配体2对增生型糖尿病视网膜病变中人视网膜微血管内皮细胞增殖、血管生成和炎症反应的影响

目的　探讨miR-106调控CC趋化因子配体2（CCL2）对增生型糖尿病视网膜病变（PDR）中人视网膜微血管内皮细胞（HRMEC）增殖、血管生成、炎症反应的影响。方法　GEO数据库筛选PDR中差异表达的miRNAs。高糖（25.0 mmol·L^-1葡萄糖,HG）诱导HRMEC建立PDR细胞模型,qRT-PCR检测miR-106和CCL2在正常葡萄糖（5.5 mmol·L^-1葡萄糖,NG）和HG培养条件下HRMEC中的表达。将PDR患者的血清外泌体做miR-106过表达处理后与HG处理的HRMEC共培养,同时干预HRMEC中CCL2的表达以探讨血清外泌体miR-106和CCL2在PDR中的功能。采用MTT法、小管形成实验和ELISA法分别检测各组HRMEC增殖、血管生成和炎症因子的表达。双荧光素酶报告实验用以验证miR-106和CCL2的靶向关系。结果　与NG组细胞中miR-106表达（1.04±0.10）、CCL2表达（1.02±0.09）相比,HG培养的HRMEC中miR-106表达（0.68±0.06）降低,CCL2表达（1.38±0.11）升高（均为P<0.05）。PDR患者血清外泌体中miR-106表达较NDR患者血清外泌体中表达降低（P<0.05）。与HG+PDR-exo+miR-NC组相比,HG+PDR-exo+miR-106 mimic组HRMEC活力降低,血管生成被抑制,细胞上清液中TNF-α、IL-1β和IL-6水平降低,而SOD、CAT和GSH水平升高（均为P<0.05）。双荧光素酶报告实验证实了CCL2是miR-106的一个靶点。与HG+PDR-exo+miR-106 mimic+oe-NC组相比,HG+PDR-exo+miR-106 mimic+oe-CCL2组HRMEC活力提高,血管生成被诱导,TNF-α、IL-1β和IL-6水平升高,而SOD、CAT和GSH水平降低（均为P<0.05）。结论　血清外泌体miR-106能够抑制CCL2表达,进而对PDR中的HRMEC增殖、血管生成和炎症反应起抑制作用。     

唾液外泌体与口腔疾病相关研究进展

唾液外泌体是指存在于唾液中的直径在30～150 nm的细胞外囊泡。随着近年来技术手段的发展,大量研究揭示唾液外泌体在多种口腔疾病的发生发展中发挥重要作用,如唾液外泌体CD9及CD81通过调控细胞粘附及运动促进肿瘤细胞转移、唾液外泌体miR-24-3p通过作用于PER1促进肿瘤细胞增殖、唾液外泌体程序性细胞死亡配体-1(programmed cell death-ligand 1,PD-L1)mRNA抑制炎症组织的破坏等,具有作为诊断口腔癌、牙周炎等口腔疾病的生物标志物的潜能。因此,唾液外泌体可作为口腔疾病潜在的预后和诊断标志物。唾液外泌体除与口腔疾病,如口腔癌、牙周炎、口腔扁平苔藓、干燥综合征等有关外,还同远处部位肿瘤如胰腺癌、肺癌等及系统性疾病如帕金森综合征、炎症性肠病等密切相关;深入研究唾液外泌体对口腔、全身系统性疾病的诊断与治疗作用,开发唾液外泌体作为疾病诊断的生物标志物的潜力具有重要意义。     

尿外泌体在膀胱尿路上皮细胞癌中的表达及临床意义

目的:探讨尿外泌体(exosome)在膀胱尿路上皮细胞癌中的表达情况及其临床意义。方法:采用超速离心法提取30例膀胱尿路上皮细胞癌患者和15例健康人群的尿外泌体。利用投射电镜观察形态,BCA法进行蛋白定量,Western blot检测尿外泌体表面分子CD9。结果:健康人群尿外泌体水平[(125.99±47.71)μg/μL]与膀胱尿路上皮细胞癌患者尿外泌体表达水平[(259.74±57.47)μg/μL]差异有统计学意义。根据肿瘤的浸润程度,非肌层浸润性膀胱癌患者尿外泌体水平与肌层浸润性膀胱癌患者尿外泌体水平差异有统计学意义。而肌层浸润性膀胱癌各期患者尿外泌体水平差异无统计学意义。结论:尿外泌体检测可能为膀胱癌的早期诊断提供一种可行的方案。     

M1表型的小胶质细胞外泌体对血脑屏障功能的影响

目的 研究M1表型的小胶质细胞外泌体（M1 microglia-derived exosome,M1-exo）对体外血脑屏障（blood-brain barrier,BBB）功能及血管内皮细胞间紧密连接蛋白表达的影响。方法 用脂多糖（lipopolysaccharide,LPS）刺激小鼠小胶质细胞来源的细胞系BV2细胞,流式技术检测其向M1型极化情况,分离提取外泌体。用小鼠脑微血管内皮细胞系b.End3细胞与原代培养的小鼠星形胶质细胞构建体外BBB模型,随机分为3组：b.End3细胞正常培养组（b.End3组）、b.End3细胞+25 μg/mL正常BV2细胞来源外泌体（BV2-derived exosome,BV2-exo）组（b.End3+BV2-exo组）、b.End3细胞+25 μg/mL M1表型的小胶质细胞来源外泌体组（b.End3+M1-exo组）。按实验分组将不同来源外泌体与BBB模型共培养,检测各组的跨膜电阻（trans-endothelial electrical resistance,TEER）、荧光黄通过率,免疫印迹法（Western blot,WB）检测紧密连接复合物蛋白Claudin-1、Occludin、ZO-1及JAM蛋白的表达。结果 ①小胶质细胞向M1型极化成功,其细胞标志物CD16/32阳性率较对照组明显升高（P=0.023）;②与M1-exo共培养后,体外BBB模型的TEER明显下降（P=0.000）,对荧光黄的透过率明显增加（P=0.000）;③与b.End3组和b.End3 + BV2-exo组相比,b.End3+M1-exo组的Claudin-1、Occludin及ZO-1蛋白的表达水平明显下降。结论 M1-exo可以破坏BBB的完整性,影响其功能。