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.

References 38

[1]	RESENDES A R, SEGALÉS J. Characterization of vascular lesions in pigs affected by porcine circovirustype 2-systemic disease[J]. Vet Pathol, 2015, 52(3):497-504.
[2]	GRAU-ROMA L, HJULSAGER C K, SIBILA M, et al. Infection, excretion and seroconversion dyna-mics of porcine circovirus type 2(PCV2) in pigs from post-weaning multisystemic wasting syndrome (PMWS) affected farms in Spain and Denmark[J]. Vet Microbiol, 2009, 135(3-4):272-282.
[3]	SHI L J, HAN H L, ZHANG S X. Virus location and lymphocyte apoptosis in lymph nodes of piglets infected with PCV-2[J]. Agric Sci China, 2008, 7(4):507-512.
[4]	DOSTER A R, SUBRAMANIAM S, YHEE J Y, et al. Distribution and characterization of IL-10-secreting cells in lymphoid tissues of PCV2-infected pigs[J]. J Vet Sci, 2010, 11(3):177-183.
[5]	KELLER S, SANDERSON M P, STOECK A, et al. Exosomes:from biogenesis and secretion to biological function[J]. Immunol Lett, 2006, 107(2):102-108.
[6]	FANG J H, ZHANG Z J, SHANG L R, et al. Hepatoma cell-secreted exosomal microRNA-103 increases vascular permeability and promotes metastasis by targeting junction proteins[J]. Hepatology, 2018, 68(4):1459-1475.
[7]	XIA T, LIAO Q, JIANG X M, et al. Long noncoding RNA associated-competing endogenous RNAs in gastric cancer[J]. Sci Rep, 2014, 4:6088.DOI:10.1038/srep06088.
[8]	SKOG J, WVRDINGER T, VAN RIJN S, et al. Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers[J]. Nat Cell Biol, 2008, 10(12):1470-1476.
[9]	NARAYANAN A, IORDANSKIY S, DAS R, et al. Exosomes derived from HIV-1-infected cells contain trans-activation response element RNA[J]. J Biol Chem, 2013, 288(27):20014-20033.
[10]	WANG G H, WANG Z Z, ZHUANG P P, et al. Exosomes carring gag/env of ALV-J possess negative effect on immunocytes[J]. Microb Pathog, 2017, 112:142-147.
[11]	ZHOU D F, XUE J W, HE S H, et al. Reticuloendotheliosis virus and avian leukosis virus subgroup J synergistically increase the accumulation of exosomal miRNAs[J]. Retrovirology, 2018, 15(1):45.DOI:10.1186/S12977-018-0427-0.
[12]	ZHANG C, YANG X, QI Q, et al. lncRNA-HEIH in serum and exosomes as a potential biomarker in the HCV-related hepatocellular carcinoma[J]. Cancer Biomark, 2018, 21(3):651-659.
[13]	HUANG B, ZHANG L L, LU M Q, et al. PCV2 infection activates the cGAS/STING signaling pathway to promote IFN-β production and viral replication in PK-15 cells[J]. Vet Microbiol, 2018, 227:34-40.
[14]	GAO W, LIU H B, YUAN J, et al. Exosomes derived from mature dendritic cells increase endothelial inflammation and atherosclerosis via membrane TNF-α mediated NF-κB pathway[J]. J Cell Mol Med, 2016, 20(12):2318-2327.
[15]	CHEN H B, ADAM A, CHENG Y F, et al. Localization and expression of heat shock protein 70 with rat myocardial cell damage induced by heat stress in vitro and in vivo[J]. Mol Med Rep, 2015, 11(3):2276-2284.
[16]	庄萍萍, 王小满, 孟薇, 等. 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[J]. 中国细胞生物学学报, 2016, 38(6):682-690.ZHUANG P P, WANG X M, MENG W, et al. The infection of reticuloendotheliosis virus changed the protein composition and immunomodulation of exosome[J]. Chinese Journal of Cell Biology, 2016, 38(6):682-690. (in Chinese)
[17]	FIGUEIREDO M M, AMORIM I F G, PINTO A J W, et al. Expression of Toll-like receptors 2 and 9 in cells of dog jejunum and colon naturally infected with Leishmania infantum[J]. BMC Immunol, 2013, 14(1):22.DOI:10.1186/1471-2172-14-22.
[18]	刘月月,吴家强,任庆海,等. 鲁中地区猪高热性疾病的病理变化及病原学调查研究[J]畜牧兽医学报,2012, 43(12):1917-1924.LIU Y Y, WU J Q, REN Q H, et al. Pathological changes and etiological study of the Swine High Fever Syndrome (SHFS) in central region of Shandong Province[J]. Acta Veterinaria et Zootechnica Sinica, 2012, 43(12):1917-1924. (in Chinese)
[19]	BECSKEI Z, NOVOSEL D, POLAČEK V, et al. Tissue distribution and tropism of PCV2 in first detected cases of porcine circovirus-associated disease (PCVAD) of wild boars in Serbia[J]. J Comp Pathol, 2015, 152(1):86.DOI:10.1016/j.jcpa.2014.10.181.
[20]	ROVIRA A, BALASCH M, SEGALÉS J, et al. Experimental inoculation of conventional pigs with porcine reproductive and respiratory syndrome virus and porcine circovirus 2[J]. J Virol, 2002, 76(7):3232-3239.
[21]	MCCULLOUGH K C, RUGGLI N, SUMMERFIELD A. Dendritic cells-at the front-line of pathogen attack[J]. Vet Immunol Immunopathol, 2009, 128(1-3):7-15.
[22]	VINCENT I E, CARRASCO C P, HERRMANN B, et al. Dendritic cells harbor infectious porcine circovirus type 2 in the absence of apparent cell modulation or replication of the virus[J]. J Virol, 2003, 77(24):13288-13300.
[23]	华利忠, 陈耿, 张书霞. 巨噬细胞介导PCV2诱导体外培养仔猪淋巴细胞凋亡的研究[J]. 南京农业大学学报, 2010, 33(3):94-98.HUA L Z, CHEN G, ZHANG S X. Study on macrophages mediated apoptosis of lymphocytes induced by PCV2 in vitro[J]. Journal of Nanjing Agricultural University, 2010, 33(3):94-98. (in Chinese)
[24]	MELO S A, LUECKE L B, KAHLERT C, et al. Glypican-1 identifies cancer exosomes and detects early pancreatic cancer[J]. Nature, 2015, 523(7559):177-182.
[25]	SANTANGELO A, IMBRUCÈ P, GARDENGHI B, et al. A microRNA signature from serum exosomes of patients with glioma as complementary diagnostic biomarker[J]. J Neurooncol, 2018, 136(1):51-62.
[26]	CLAYTON A, HARRIS C L, COURT J, et al. Antigen-presenting cell exosomes are protected from complement-mediated lysis by expression of CD55 and CD59[J]. Eur J Immunol, 2003, 33(2):522-531.
[27]	TUCCI M, PASSARELLI A, MANNAVOLA F, et al. Serum exosomes as predictors of clinical response to ipilimumab in metastatic melanoma[J]. Oncoimmunology, 2018, 7(2):e1387706.DOI:10.1080/2162402X.2017.1387706.
[28]	LÄSSER C, ELDH M, LÖTVALL J. Isolation and characterization of RNA-containing exosomes[J]. J Vis Exp Jove, 2012(59):3037.DOI:10.3791/3037.
[29]	WANG T, FANG L R, ZHAO F W, et al. Exosomes mediate intercellular transmission of porcine reproductive and respiratory syndrome virus[J]. J Virol, 2018, 92(4):e01734-17.
[30]	Longatti A. The dual role of exosomes in hepatitis A and C virus transmission and viral immune activation[J]. Viruses, 2015, 7(12):6707-6715.
[31]	CHAHAR H S, BAO X Y, CASOLA A. Exosomes and their role in the life cycle and pathogenesis of RNA viruses[J]. Viruses, 2015, 7(6):3204-3225.
[32]	COSSET F L, DREUX M. HCV transmission by hepatic exosomes establishes a productive infection[J]. J Hepatol, 2014, 60(3):674-675.
[33]	JAWORSKI E, NARAYANAN A, VAN DUYNE R, et al. Human T-lymphotropic virus type 1-infected cells secrete exosomes that contain Tax protein[J]. J Biol Chem, 2014, 289(32):22284-22305.
[34]	EL-SAGHIR J, NASSAR F, TAWIL N, et al. ATL-derived exosomes modulate mesenchymal stem cells:potential role in leukemia progression[J]. Retrovirology, 2016, 13(1):73.DOI:10.1186/S12977-0160-0307-4.
[35]	FENG Z D, HENSLEY L, MCKNIGHT K L, et al. A pathogenic picornavirus acquires an envelope by hijacking cellular membranes[J]. Nature, 2013, 496(7445):367-371.
[36]	GOULD S J, BOOTH A M, HILDRETH J E K. The Trojan exosome hypothesis[J]. Proc Natl Acad Sci U S A, 2003, 100(19):10592-10597.
[37]	KRUISBEEK A M. Age-related changes in ConA-and LPS-induced lymphocyte transformation. I. Effect of culture conditions on mitogen responses of blood and spleen lymphocytes from young and aged rats[J]. Mech Ageing Dev, 1976, 5:125-138.
[38]	MANDRIOLI L, SARLI G, PANARESE S, et al. Apoptosis and proliferative activity in lymph node reaction in postweaning multisystemic wasting syndrome (PMWS)[J]. Vet Immunol Immunopathol, 2004, 97(1-2):25-37.

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

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References 38

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	CHEN Songbiao, LIU Feifei, SHANG Ke, YU Zuhua, HE Lei, WEI Ying, CHEN Jian, ZHANG Chunjie, CHENG Xiangchao, DING Ke. Molecular Mechanism of the “Battle” between Virus Infection and Host Antiviral Immunity-Apoptosis, Necroptosis and Pyroptosis [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(1): 59-70.
[2]	WANG Xinxin, LIN Shumei, ZHAO Dongdong, WANG Xuesheng. Role of Exosomes Secreted by Alveolar Epithelial Cells in Regulating Macrophage Polarization in Acute Lung Injury [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(1): 71-78.
[3]	DUAN Xiangru, KANG Jia, YANG Ruochen, SHAN Xinyu, LI Taichun, ZHAO Wen, ZHANG Yingjie, LIU Yueqin. Effect of L-cysteine on Proliferation, Apoptosis and the Secretion of Steroid Hormone in Ovine Ovarian Granulosa Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(1): 179-191.
[4]	LIANG Kaixin, ZHONG Haiwen, SONG Changxu, YANG Huaqiang, HUANG Sixiu, XU Zheng. Study on the Effect of SYNGR2 on PCV2 Replication [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(9): 3824-3835.
[5]	ZHANG Wanfeng, ZHAO Tianzhi, LI Jiao, YOU Ziwei, YANG Yang, CAI Chunbo, GAO Pengfei, CAO Guoqing, GUO Xiaohong, LI Bugao. Study on NR2F2 Gene Regulating Proliferation and Apoptosis of Porcine PK15 Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3242-3251.
[6]	SONG Meijun, HAO Kexing, HAI Siyu, CHEN Yan, WANG Jing, HU Guangdong. Effects of SRIF-14 on Proliferation and Apoptosis of Endometrial Epithelial Cells in Sheep [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3325-3334.
[7]	XU Xi, YANG Baigao, ZHANG Hang, FENG Xiaoyi, HAO Haisheng, DU Weihua, ZHU Huabin, ZHANG Peipei, ZHAO Xueming. Effects of NMN on Lipid Droplet Content and Cryopreservation Effect of Bovine Oocytes [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3348-3357.
[8]	WANG Wanjie, CHEN Nanzhu, ZOU Huiying, ZHOU Xinyi, HAO Haisheng, PANG Yunwei, ZHU Huabin, ZHAO Xueming, YU Dawei, DU Weihua. Effects of Histone Methyltransferase ASH1L Overexpression on Proliferation and Apoptosis of Bovine Cumulus Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3358-3368.
[9]	SHEN Tong, WANG Mengjie, WU Hua, LI Jinming, WANG Shuo, WU Xiaoqing, CHEN Xinlei, XING Qianwen, LIU Bo. Effect of Lycium ruthenicum Murray Anthocyanin on Hypoxia-induced Apoptosis in H9c2 Rat Cardiomyocytes [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3490-3499.
[10]	ZHANG Peng, WANG Mingxiu, JING Kemin, PENG Wei, TIAN Yuan, LI Yuqian, FU Changqi, SHU Shi, ZHONG Jincheng, CAI Xin. Abnormal Expression of FGFs/FGFRs and Their Mediated Signaling Pathway Genes Affect the Proliferative Activity of Undifferentiated Spermatogonia in Cattleyak [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(7): 2886-2897.
[11]	AN Qi, YU Jialin, WU Xiaoling, DENG Guangcun. Regulation of BCG-induced Apoptosis in Murine Macrophages by Glutamine [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(7): 3054-3063.
[12]	ZHAO Yangfei, YU Yanghuan, WANG Jinming, ZHANG Jianhai, SUN Zilong, NIU Ruiyan, WANG Jundong. Effects of IL-17A Knockout on Fluoride-Induced Hepatic Inflammation and Hepatocyte Apoptosis [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(7): 3108-3117.
[13]	ZHAO Donghao, YUAN Meng, MA Kaiteng, DUAN Zhuo, ZHU Yixin, TANG Fang, HAN Keguang, HUO Nairui. Chelating Role of Sheep Bone Collagen Peptide to Cadmium and Its Protection Role against Liver Injuries Induced by Cadmium in Chickens [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(6): 2641-2652.
[14]	XU Tiantian, ZHANG Tongtong, WANG Meng, WANG Xin. Transcription Factor Foxq1 Affects the Proliferation of Hair Follicle Stem Cells in Cashmere Goats via WNT/β-catenin Signaling Pathway [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(6): 2653-2661.
[15]	ZHU Junru, WEI Wei, PEI Dangshuai, ZHANG Fenque, DUAN Yu, GUO Yongfeng, JIANG Yue, XIA Shuli, HAN Jing, HOU Jinxing, AN Xiaopeng. Effects of PDCD4 on the Apoptosis of Dairy Goat Mammary Epithelial Cells and the Synthesis of β-casein and TG [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(4): 1429-1440.