猪圆环病毒2型感染PK-15细胞中外泌体的鉴定及其对细胞增殖和凋亡的影响

旨在分离感染PCV2的PK-15细胞分泌的外泌体，探讨其在PCV2感染淋巴细胞中的作用。提取PCV2感染PK-15细胞上清液中的外泌体，利用透射电镜观察外泌体形态、纳米颗粒跟踪分析测定外泌体粒径、Western blot检测外泌体标记蛋白分子（CD81、TSG101）；PKH67标记外泌体后检测细胞对外泌体的摄取；提取PCV2-外泌体基因组，检测PCV2 Rep和Cap基因；利用间接免疫荧光试验检测PCV2-外泌体感染PK-15细胞后PCV2病毒定位；通过绝对定量PCR检测PCV2-外泌体对淋巴细胞的感染率；利用CCK-8法检测淋巴细胞增殖；利用Annexin V-FITC/PI检测淋巴细胞凋亡率。透射电镜和外泌体粒径分析结果显示，PK-15细胞分泌的外泌体为双层膜的囊泡，直径30~200 nm；Western blot结果显示PK-15细胞分泌的外泌体存在特异性标记蛋白CD81和TSG101；外泌体摄取试验结果显示PCV2-外泌体能够被细胞摄取；PCR结果表明PCV2-外泌体基因组中含有PCV2 Rep和Cap基因；间接免疫荧光结果显示，与PCV2直接感染相比，PCV2-外泌体感染的淋巴细胞中，PCV2病毒拷贝显著升高，差异明显（P<0.01），外泌体裂解后感染淋巴细胞能力与PCV2病毒无显著差异；PCV2-外泌体可显著抑制淋巴细胞增殖（P<0.01或P<0.05）；PCV2-外泌体显著提高淋巴细胞凋亡率。PCV2感染PK-15细胞的外泌体中携带病毒基因，感染淋巴细胞后使细胞增殖降低和细胞凋亡增加。

Identification of Exosomes in Porcine Circovirus Type 2 Infected PK-15 Cells and Its Effects on Cell Proliferation and Apoptosis

The objective of this study was to isolate the exosomes of PK-15 cells infected with PCV2 and explore its role in lymphocyte infected with PCV2. The exosomes in the supernatant of PK-15 were extracted. We observed the morphology of the exosomes by transmission electron microscopy (TEM), measured the particle size of the exosomes by nanoparticle tracking analysis (NTA), and detected their molecular markers CD81 and TSG101 by Western blot. PKH67 labeled exosomes were used to detect the uptake of exosomes by cells. The PCV2-exosome genome was extracted and PCV2 Rep and Cap genes were detected. Indirect immunoinfluscent assay (IFA) was used to detect the localization of the PCV2 in PK-15 cells infected with PCV2-exosome. The infection rate of PCV2-exosome on lymphocyte was detected by absolute quantitative PCR. Lymphocyte proliferation were detected by using CCK-8 method. The apoptosis rate of lymphocytes was detected by Annexin V-FITC/PI. The results showed that exosomes had bilayer membrane vesicles with a diameter of 30-200 nm by using TEM and NTA. CD81 and TSG101, two exosomal protein markers were expressed in the purified exosomes. The results of the exosome uptake experiment showed that exosomes could be taken by PK-15 cells. The PCR results showed that PCV2-exosome genome contained PCV2 Rep and Cap genes. The IFA results showed that compared with PCV2 direct infection, PCV2 copies in lymphocytes infected with PCV2 exosomes were significantly higher (P<0.01). There was no significant difference between PCV2 virus and PCV2-exosome lysate on the lymphocyte infection ability. The lymphocyte proliferation was significantly inhibited by PCV2-exosome (P<0.01 or P<0.05). The lymphocyte apoptosis was significantly increased by PCV2-exosome. These findings suggest that the exosomes purified from PCV2 infected PK-15 cells contained viral genes, which can reduce cell proliferation and increase cell apoptosis in lymphocyte.