网状内皮增生症病毒感染改变外泌体蛋白质组成和免疫调节功能

The Infection of Reticuloendotheliosis Virus Changed the Protein Composition and Immunomodulation of Exosome

采用间接免疫荧光、透射电镜观察、Westernblot、质谱分析、酶联免疫吸附试验（enzyme-linked immunosorbent assay，ELISA）和流式细胞术分析技术检测了网状内皮增生症病毒（reticuloendotheliosis virus，REV）感染DF－1细胞的情况、感染细胞分泌外泌体（exosome）的形态大小、标志蛋白质水平以7LREV对外泌体蛋白质组成和免疫功能的影响。结果显示，接毒成功的DF－1细胞胞质中有明显的绿色荧光出现。提取的外泌体呈杯状，平均直径大小在50～100nm，表达标志蛋白质Hsp70和Tsg101。与正常未感染细胞相比，REV感染细胞分泌的外泌体共存在58种表达水平差异的蛋白质。其中上调的46种，包括REV的env蛋白gp90和gag—pol在内的4种蛋白质，下降的12种。这些外泌体同时具有明显的促进脾细胞分泌IL－10的作用；对CD4＋T和CD8＋T细胞均具有明显的抑制性。REV感染改变了外泌体的蛋白质组成，尤其是携带了REV的重要成分，该成分与引起的外泌体抑制性免疫调节作用密切相关。

The indirect immunofluorescence, spectrometry analysis, ELISA and flow cytometric were transmission electron microscopy, Western blot, mass used to investigate the reticuloendotheliosis virus （REV） infected DF-1, the morphology and biomarkers of exosome and the effects of REV on protein composition and immunomodulation of exosome from infected DF-1 cells （Exo-R）. The results demonstrated that green fluorescence was observed in the cytoplasm of infected DF-1 ceils. Isolated exosome was up to 50-100 nm in diameter, with a cup-shape structure, and expressed marker proteins Hsp70 and Tsgl01. Compared with exosome from uninfected cells, 58 kinds of different proteins were detected in Exo-R with up-regulated 46 kinds containing env protein gp90 and gag-pol of REV, and down-regulated 12 kinds. Furthermore, the secretion of IL-10 was increased from splenocytes treated by Exo-R. The number of CD4＋T and CD8＋T was also significantly decreased. The infection of REV induced the protein composition change of exosome,especially those important proteins carried by REV, which was associated with the immunosuppressive effects induced by exosome.