| Isolation, BODIPY Labeling and Uptake of Exosomes in Hepatic Stellate Cells
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Author:
Date:
2017-12-05
[Abstract] Exosomes have emerged as an important mediator of intercellular communication. They are present in extracellular milieu and therefore, easily accessible by neighboring or distant cells. They carry mRNA, microRNAs and proteins within their vesicles and once internalized by recipient cells; they can modulate multiple signaling pathways with pleiotropic effects from inducing antiviral state to disease progression. We have previously shown that hepatitis C virus (HCV) infected hepatocytes or hepatoma cells harboring genome-length replicon secrete exosomes in culture supernatants. These exosomes are taken up by hepatic stellate cells (HSC) and activate them to induce fibrosis during HCV infection. Here, we describe detailed protocols for exosomes isolation and uptake of BODIPY labeled exosomes ...
[摘要] 外来体已经成为细胞间通讯的重要介质。 它们存在于细胞外环境中，因此可以被邻近或远处的细胞轻易获得。 它们在囊泡内携带mRNA，microRNA和蛋白质，并一度被受体细胞内化; 它们可以通过诱导抗病毒状态向疾病进展调节多种信号传导途径，具有多效性。 我们以前已经显示，携带基因组长度复制子的丙型肝炎病毒（HCV）感染的肝细胞或肝癌细胞在培养上清液中分泌外泌体。 这些外来体被肝星状细胞（HSC）摄取，并在HCV感染期间激活它们以诱导纤维化。 在这里，我们描述了外来体分离和肝星状细胞摄取BODIPY标记的外来体的详细的协议。
【背景】外来体是直径为40-150nm的小的膜结合的细胞外囊泡。已在所有体液中鉴定出它们，包括尿，羊水，血清，唾液，母乳，脑脊液，鼻分泌物和组织培养的细胞系的上清液中。外泌体通过转移微小RNA（mRNA），mRNA和蛋白质来介导细胞与细胞之间的通讯。 其可被邻近或远处的细胞摄取，并随后促进受体细胞中的信号传导（Schorey等人，2015）。外泌体富含蛋白质，包括四跨膜蛋白家族成员（CD9，CD81，CD63），热休克蛋白（Hsp60，Hsp70，Hsp90）和多泡体蛋白（annexins，RabGTPases和内体分选复合物，蛋白质）。最近的研究表明，外来体可以影响免疫应答（Li等人，2013; ...
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| miRNA Characterization from the Extracellular Vesicles
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Author:
Date:
2017-02-20
[Abstract] Cancer cells and cancer associated stromal cells co-evolve secrete extracellular vesicles to the surrounding regions and regulate several processes involved in cancer metastasis. miRNAs have been known to be mediators of cancer progression and metastasis. miRNAs consist of short noncoding RNA. miRNAs are stable in extracellular fluids such as serum, plasma and urine. miRNAs are secreted by cells in normal and diseased conditions. miRNAs signatures have been identified specific to certain disease conditions. Therefore they are valuable biomarkers for different diseases. In our study we identified certain miRNAs, miR-409-3p and miR-409-5p, which were secreted by activated stromal fibroblast cells and were taken up by cancer cells to induce explosive tumor growth, through activation of ...
[摘要] 癌细胞和癌症相关的基质细胞将分泌的细胞外囊泡共同进入周围区域并调节参与癌症转移的几个过程。已知miRNA是癌症进展和转移的介质。 miRNA由短的非编码RNA组成。 miRNA在细胞外液体如血清，血浆和尿液中是稳定的。 miRNA在正常和患病条件下由细胞分泌。已经鉴定了特定疾病条件的miRNAs特征。因此，它们是不同疾病的宝贵生物标志物。在我们的研究中，我们确定了某些miRNAs，miR-409-3p和miR-409-5p，其由激活的基质成纤维细胞分泌并被癌细胞摄取以诱导爆发性肿瘤生长，通过激活癌细胞的上皮至间质转化细胞。在这里，我们描述了一种在细胞外囊泡中测定miRNA（miR-409-3p和miR-409-5p）的方法，其由表达miR-409的前列腺癌基质细胞分泌。在该方法中，使用来自间质成纤维细胞的条件培养基来提取囊泡部分。从囊泡部分纯化RNA，并使用实时PCR测定将特异性miRNA逆转录并定量。
背景 miRNA是大小为20-23个核苷酸的短非编码RNA。已经在组织和体液中检测到miRNA。已经使用Northern印迹和定量实时PCR测定了miRNA表达水平。 ...
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