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

doi:10.11843/j.issn.0366-6964.2020.07.021

Abstract

Abstract: 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.

Key words: exosome, PCV2, lymphocyte, cell proliferation, apoptosis

CLC Number:

S852.659.2

CHEN Hongbo, DUAN Dianning, HU Yao, YANG Runze, HONG Qixiang, LI Yu, QIU Longxin, YANG Xiaoyan. Identification of Exosomes in Porcine Circovirus Type 2 Infected PK-15 Cells and Its Effects on Cell Proliferation and Apoptosis[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(7): 1688-1698.

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

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