随机光学重构显微镜在外泌体观察中的应用

目的探讨随机光学重构显微镜（STORM）在外泌体观察中的应用价值,并介绍外泌体在随机光学重构显微镜下的成像原理及方法。方法实验中在肾性甲旁亢患者原代培养的甲状旁腺细胞的培养液上清中加入EXOQUICK-TC外泌体沉淀剂来获取细胞培养上清中的外泌体,将外泌体膜表面的特异性跨膜蛋白CD63作免疫荧光标记后利用随机光学重构显微镜对外泌体行超高分辨率成像并测量外泌体直径。结果实验中随机光学重构显微镜成功的对继发性甲旁亢甲状旁腺细胞原代培养液上清中的外泌体行单分子定位、直径测量及超高分辨率成像。结论因随机光学重构显微镜具有独特的光学特性并突破了光学衍射极限的限制,较传统光学显微镜,能获得20~50 nm的分辨率,可对外泌体行单分子精确定位、直径测量、超高分辨率成像。基于STORM的成像优势,相信STORM及其他超高分辨率成像技术将在外泌体及外泌体参与的生物学过程的研究中发挥重要作用。     

外泌体在肿瘤体外诊断中的临床研究和应用前景

外泌体由于富含生物信息分子,提供了潜在的生物标志物,在肿瘤体外诊断中显示出良好的临床应用前景。阐述了外泌体的功能、在体外诊断中的优势及检测方法,并深入探讨了外泌体在肺癌、肠癌、卵巢癌、胃癌、胰腺癌、膀胱癌、骨与软组织肉瘤等肿瘤体外诊断中的价值和临床获批情况,展望了外泌体在体外诊断领域的发展趋势。     

Exosomes play roles in sequential processes of tumor metastasis

Overwhelming evidence demonstrates that exosomes, a series of biologically functional small vesicles of endocytic origin carrying a variety of active constituents, especially tumor-derived exosomes, contribute to tumor progression and metastasis. This review focuses on the specific multifaceted roles of exosomes in affecting sequenced four crucial processes of metastasis, through which cancer cells spread from primary to secondary organs and finally form macroscopic metastatic lesions. First, exosomes modulate the primary tumor sites to assist cancer growth and dissemination. In this part, five main biological events are reviewed, including the transfer of oncogenic constituents, the recruitment and activation of fibroblasts, the induction of angiogenesis, immunosuppression and epithelial-mesenchymal transition (EMT) promotion. In Step 2, we list two recently disclosed mechanisms during the organ-specific homing process: the exosomal integrin model and exosomal epidermal growth factor receptor (EGFR)/miR-26/hepatocyte growth factor (HGF) model. Subsequently, Step 3 focuses on the interactions between exosomes and pre-metastatic niche, in which we highlight the specific functions of exosomes in angiogenesis, lymphangiogenesis, immune modulation and metabolic, epigenetic and stromal reprogramming of pre-metastatic niche. Finally, we summarize the mechanisms of exosomes in helping the metastatic circulating tumor cells escape from immunologic surveillance, survive in the blood circulation and proliferate in host organs.     

Role of exosomes in the immune microenvironment of ovarian cancer

Exosomes are excretory vesicles that can deliver a variety of bioactive cargo molecules to the extracellular environment. Accumulating evidence demonstrates exosome participation in intercellular communication, immune response, inflammatory response and they even play an essential role in affecting the tumor immune microenvironment. The role of exosomes in the immune microenvironment of ovarian cancer is mainly divided into suppression and stimulation. On one hand exosomes can stimulate the innate and adaptive immune systems by activating dendritic cells (DCs), natural killer cells and T cells, allowing these immune cells exert an antitumorigenic effect. On the other hand, ovarian cancer-derived exosomes initiate cross-talk with immunosuppressive effector cells, which subsequently cause immune evasion; one of the hallmarks of cancer. Exosomes induce the polarization of macrophages in M2 phenotype and induce apoptosis of lymphocytes and DCs. Exosomes further activate additional immunosuppressive effector cells (myeloid-derived suppressor cells and regulatory T cells) that induce fibroblasts to differentiate into cancer-associated fibroblasts. Exosomes also induce the tumorigenicity of mesenchymal stem cells to exert additional immune suppression. Furthermore, besides mediating the intercellular communication, exosomes carry microRNAs (miRNAs), proteins and lipids to the tumor microenvironment, which collectively promotes ovarian cancer cells to proliferate, invade and tumors to metastasize. Studying proteins, lipids and miRNAs carried by exosomes could potentially be used as an early diagnostic marker of ovarian cancer for designing treatment strategies.     

血清外泌体Glypican-1在乳腺癌中的表达水平及临床应用价值

目的 探讨乳腺癌血清外泌体Glypican-1（GPC-1）的表达水平及其临床应用价值。 方法 选择河北医科大学第四医院确诊的乳腺癌48例和乳腺良性占位18例，同期健康体检者17名作为健康对照组；采用聚乙二醇8 000沉淀法提取血清外泌体，使用透射电镜和蛋白印迹对外泌体形态和分子表型进行鉴定；Western blot检测各组血清外泌体GPC-1表达水平；采用受试者工作特征（receiver operating characteristic，ROC）曲线评价血清外泌体GPC-1对乳腺癌的诊断效能；以最佳截断值0.961将乳腺癌组分为GPC-1阳性组和GPC-1阴性组，分析其与临床特征的关系；采用Wilcoxon秩和检验分析乳腺癌组治疗前后血清外泌体GPC-1表达变化；以病理诊断为金标准，采用Kappa一致性检验评价血清外泌体GPC-1、血清肿瘤标志物、超声、钼靶射线四种诊断方法对乳腺癌的诊断价值。 结果 乳腺癌组血清外泌体GPC-1表达水平［1.065(0.501)］高于乳腺良性占位组［0.652(0.117)］和健康对照组［0.625(0.139)］，差异有统计学意义（H=32.051，P＜0.05）；ROC曲线下面积为0.900（95%CI:0.833~0.967），最佳截断值0.961，能够有效筛查乳腺癌患者；乳腺癌组中GPC-1阳性在不同TNM分期、淋巴结浸润和Ki-67高表达组间差异有统计学意义（P均＜0.05）；乳腺癌组治疗前血清外泌体GPC-1表达水平［1.065(0.501)］高于治疗20 d后血清外泌体GPC-1表达水平［0.844(0.195)］，差异有统计学意义（Z=856.0，P=0.006）；血清外泌体GPC-1检测方法的Kappa值为0.485>0.4，与病理检查比较一致性中等，且优于其他三种检测方法。 结论 乳腺癌组血清外泌体GPC-1表达上调,且与多个不良临床特征相关，并在治疗后显著下降，具备作为乳腺癌诊断和治疗效果评价指标的潜力。     

Characterization of a purified exosome product and its effects on canine flexor tenocyte biology

Flexor tendon injuries and tendinopathy are very common but remain challenging in clinical treatment. Exosomes-based cell-free therapy appears to be a promising strategy for tendon healing, while limited studies have evaluated its impacts on tenocyte biology. The objective of this study was to characterize a novel purified exosome product (PEP) derived from plasma, as well as to explore its cellular effects on canine tenocyte biology. The transmission electron microscope revealed that exosomes of PEP present cup-shaped structures with the diameters ranged from 80 to 141 nm, and the NanoSight report presented that their size mainly concentrated around 100 nm. The enzyme-linked immunosorbent assay kits analysis showed that PEP was positive for CD63 and AChE expression, and the cellular uptake of exosomes internalized into tenocyte cytoplasm was observed. The cell growth assays displayed that tenocyte proliferation ability was enhanced by PEP solution in a dose-dependent manner. Tenogenic phenotype was preserved as is evident by that tendon-related genes expression (SCX, COL1A, COL3A1, TNMD, DCN, and MKX) were expressed insistently in a high level, while tenocytes were treated with 5% PEP solution. Furthermore, migration capability was maintained and total collagen deposition was increased. More interesting, dexamethasone-induced cellular apoptosis was attenuated during the incubation of tenocytes with a 5% PEP solution. These findings will provide the basic understandings about the PEP, and support the potential use of this biological strategy for tendon healing.     

Quantitative changes in the secretion of exosomes from keratinocytes homeostatically regulate skin pigmentation in a paracrine manner

The content and distribution of melanin in the epidermis determines the wide variety of skin colors associated with ethnic/racial diversity. Although it was previously reported that qualitative changes in keratinocyte-derived exosomes regulate melanocyte pigmentation in vitro, their practical involvement, especially in skin color development in vivo, has remained unclear. To address this unexplained scientific concern, the correlation of epidermal exosomes isolated from human skin tissues with melanosomal protein expression levels was demonstrated in this study for the first time. After confirming the quantitative effect of human keratinocyte-derived exosomes on human melanocyte activation, even in the absence of ultraviolet B (UV-B) exposure, the impact of exosomes secreted from UV-B-irradiated keratinocytes on melanogenesis was consistently detected, which suggests their constitutive role in regulating cutaneous pigmentation. Additionally, both a specific exosome secretion inducer and a suppressor were consistently found to significantly control melanin synthesis in a co-culture system composed of keratinocytes and melanocytes as well as in an ex vivo skin culture system. These results suggest that quantitative changes, in addition to already known qualitative changes, in exosomes secreted from human epidermal keratinocytes homeostatically regulate melanogenic activity in a paracrine manner, which leads to skin color determination.     

Local oncolytic adenovirotherapy produces an abscopal effect via tumor-derived extracellular vesicles

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