PDF(Pubmed)
Abstract：
To mediate intercellular communication, cells produce extracellular vesicles (EVs). These EVs transport many biomolecules such as proteins, nucleic acids, and lipids between cells and regulate pathophysiological actions in the recipient cell. However, EVs and virus particles produced from virus-infected cells are of similar size and specific gravity; therefore, the separation and purification of these two particles is often controversial. When analyzing the physiological functions of EVs from virus-infected cells, the presence or absence of virus particle contamination must always be verified. The human T-cell leukemia virus type 1 (HTLV-1)-infected cell line, MT-2, produces EVs and virus particles. Here, we validated a method for purifying EVs from MT-2 cell culture supernatants while avoiding HTLV-1 viral particle contamination. EV fractions were collected using a combination of immunoprecipitation with Tim-4, which binds to phosphatidylserine, and polymer precipitation. The HTLV-1 viral envelope protein, gp46, was not detected in the EV fraction. Proteomic analysis revealed that EV-constituted proteins were predominant in this EV fraction. Furthermore, the EVs were found to contain the HTLV-1 viral genome. The proposed method can purify EVs while avoiding virus particle contamination and is expected to contribute to future research on EVs derived from HTLV-1-infected cells.
摘要：
为了调解细胞间的通讯，细胞产生细胞外囊泡(EV)。这些电动汽车运输许多生物分子，如蛋白质，核酸,和细胞之间的脂质并调节受体细胞的病理生理作用。然而,由病毒感染的细胞产生的EV和病毒颗粒具有相似的大小和比重；因此，这两种颗粒的分离和纯化经常是有争议的。当从病毒感染的细胞分析电动汽车的生理功能时，必须始终验证是否存在病毒颗粒污染。人类T细胞白血病病毒1型（HTLV-1）感染细胞系，MT-2产生电动汽车和病毒颗粒。这里,我们验证了一种从MT-2细胞培养上清液中纯化EV的方法,同时避免了HTLV-1病毒颗粒污染.使用与磷脂酰丝氨酸结合的Tim-4免疫沉淀的组合收集EV级分，和聚合物沉淀。HTLV-1病毒包膜蛋白，在EV分数中未检测到gp46。蛋白质组学分析显示，EV组成的蛋白质在该EV部分中占主导地位。此外，EV被发现含有HTLV-1病毒基因组。所提出的方法可以纯化电动汽车，同时避免病毒颗粒污染，预计将有助于未来对来自HTLV-1感染细胞的电动汽车的研究。
