猪乳外泌体对猪流行性腹泻病毒的抑制作用

doi:10.13560/j.cnki.biotech.bull.1985.2021-0112

摘要/Abstract

摘要：

本文旨在探索猪乳外泌体（exosome）对仔猪小肠上皮细胞（IPEC-J2）中PEDV的抑制作用。试验采用结晶紫染色及MTT方法分别测定细胞活性,qRT-PCR和Western blot分别测定病毒及细胞相关基因和蛋白的表达水平。结果显示,猪乳exosome显著抑制PEDV病毒对IPEC-J2细胞的感染力,细胞存活率和活性显著升高（P<0.05）;极显著下调感染PEDV后细胞内及细胞上清中病毒的M、N、ORF3、Spike、RNA polymerase和E等基因表达量（P<0.01）;猪乳exosome组细胞内PEDV病毒N蛋白及IPEC-J2细胞内凋亡CLDN1 蛋白表达量显著下调;猪乳exosome三种不同处理方式下,处理方式2（杀灭）和3（修复）分别对免疫相关IFN-a和IRF3基因表达影响不明显（P>0.05）,其他各处理组对猪氨肽酶N（pAPN）表达量极显著下调（P<0.01）,而对NF-κB基因的表达无显著影响（P>0.05）;以上结果提示,猪乳exosome在3种不同的处理方式下均能降低PEDV对仔猪肠道上皮IPEC-J2细胞的感染能力,推测其机制可能一方面通过抑制病毒感染或复制相关的基因降低感染性,另一方面通过降低细胞内凋亡或提高免疫相关基因的表达增强细胞对病毒的抵抗能力,从而达到对仔猪肠道上皮细胞的保护作用。

关键词: 猪乳外泌体, 仔猪流行性腹泻病毒, 抑制作用

Abstract:

This work aims to explore the effects of porcine milk-derived exosome on porcine epidemic diarrhea virus in IPEC-J2 cells. We adapted crystal violet and MTT methods to measure cytoactive,and real-time-PCR and Western Blot to measure the expressions of PEDV virus and cell relative genes and proteins,respectively. The results showed that the porcine milk-derived exosome significantly inhibited the infectivity of PEDV virus to IPEC-J2 cells,and the cell survival rate and activity significantly increased（P<0.05）. The M,N,ORF3,Spike,RNA polymerase and E genes of the virus inside the infected cells with PEDV and cell supernatant were extremely down-regulated（P<0.01）. The PEDV virus N protein and IPEC-J2 cells’ apoptotic CLDN1 protein expressions were significantly inhibited by the porcine milk-derived exosome. Under different treatments,treatment 2（kill）and 3（repair）had no effect on the expressions of immune-related IFN-a and IRF3 genes respectively（P>0.05）,and the other treatments extremely down-regulated the expression of pAPN（P<0.01）,but had no significant effect on the expression of NF-κB gene（P>0.05）. All above results suggest that porcine milk-derived exosome under 3 different treatments may reduce the infectivity of the PEDV to the intestinal epithelial IPEC-J2 cells in piglets. It is speculated that its mechanism may reduce infectivity by inhibiting viral infection or replication-related genes on the one hand,and on the other hand,or enhance the resistance of cells to viruses by reducing intracellular apoptosis or increasing the expressions of immune-related genes t,so as to achieve the protective effect of piglet intestinal epithelial cells,but the specific regulation mechanism needs to be further investigated.

Key words: porcine milk-derived exosome, porcine epidemic diarrhea virus, inhibitory effect

陈婷, 谢梅英, 魏立民, 欧阳坤, 程晓, 张永亮. 猪乳外泌体对猪流行性腹泻病毒的抑制作用[J]. 生物技术通报, 2021, 37(12): 141-150.

CHEN Ting, XIE Mei-ying, WEI Li-min, OUYANG Kun, CHENG Xiao, ZHANG Yong-liang. Inhibitory Effects of Porcine Milk-derived Exosome on Porcine Epidemic Diarrhea Virus[J]. Biotechnology Bulletin, 2021, 37(12): 141-150.

图/表 8

表1 引物序列

Table 1 Primer sequences

Gene	Primer sequence（5'-3'）
ORF3-F	CGGGCTTCGTTTAGTCTGCT
ORF3-R	GATGTAATGGTCGCCACCTTCT
N protein-F	AAAACGGGTGCCATTATCTCT
N protein-R	CCATTTGCTGGTCCTTATTCC
M protein-F	TCCCGTTGATGAGGTGATTG
M protein-R	AAGGATGCTGAAAGCGAAAA
RNA polymerase-F	GACCGCAGGCTATCCTTTGA
RNA polymerase-R	GCTCTATCGCACTTTGGGTAATC
spike protein-F	GATGACATTTATTCCCGACTGG
spike polymerase-R	GCTGAGATTGCGATTTGACG
S protein-F	GCAGTAATTCCTCAGATCCTC
S protein-R	GTAGTGTCAGATGCAATGAGG
E protein-F	GGCTCAGAGCAAGAGAGGTATCC
E protein-R	GGTCTCAAACATGATCTGAGTCATCT
NF-Kb-F	CCAGCCCTATCCCTTTACGC
NF-Kb-R	GCCTCTGTCAGTGTCCCTTCC
IFN-F	GCAACCAGGTCCAGAAGGC
IFN-R	GACCTAGTCGTCGAGTCCC
IRF-3-F	GCACTCACCGTCGTCATTC
IRF-3-R	CAGAAAAGGCCGTGGAAATA
p-APN-F	GGATTGTTCCCATCTCATCTATT
p-APN-R	TTTTGGCGTAGCCTGCT

图1 结晶紫染色细胞形态学观察

Fig. 1 Crystal violet staining and cell morphology observation

图2 细胞存活力统计

Fig. 2 Cell survival activity statistics

图3 MTT检测细胞活力

Fig. 3 Cell activity detected by MTT

图4 猪乳exosome对PEDV相关基因在IPEC-J2细胞内表达的影响

Fig. 4 Expressions of PEDV-related genes treated with porcine milk-derived exosome in IPEC-J2 cells

图5 猪乳exosome对PEDV相关基因在IPEC-J2细胞上清中表达的影响

Fig. 5 Expressions of PEDV-related genes treated with porcine milk-derived exosome in IPEC-J2 cells supernatant

图6 PEDV病毒及细胞凋亡相关蛋白表达

Fig. 6 Expressions of apoptosis-related and PEDV-related proteins

图7 猪乳exosome对IPEC-J2细胞免疫相关基因表达的影响

Fig. 7 Expressions of immune-related genes treated with porcine milk-derived exosome in IPEC-J2 cells

参考文献 36

[1]	郭世栋, 穆娟. 仔猪流行性腹泻的诊治[J]. 饲料与畜牧, 2017（20）:57, 59.
	Guo SD, Mu Mu. Diagnosis and treatment of porcine epidemic diarrhea[J]. Feed and Husbandry, 2017（20）:57, 59.
[2]	郭效珍. PEDV感染vero细胞的蛋白质组学分析及诱导细胞自噬的机制研究[D]. 武汉:华中农业大学, 2017.
	Guo XZ. Proteomics and molecular mechanisms of autophagy in PEDV-infected vero cells[D]. Wuhan:Huazhong Agricultural University, 2017.
[3]	van Niel G, Porto-Carreiro I, Simoes S, et al. Exosomes:a common pathway for a specialized function[J]. J Biochem, 2006, 140(1):13-21.
[4]	Conde-Vancells J, Rodriguez-Suarez E, Gonzalez E, et al. Candidate biomarkers in exosome-like vesicles purified from rat and mouse urine samples[J]. Proteomics Clin Appl, 2010, 4(4):416-425. doi: 10.1002/prca.v4:4 URL
[5]	Théry C, Amigorena S, Raposo G, et al. Isolation and characterization of exosomes from cell culture supernatants and biological fluids[J]. Curr Protoc Cell Biol, 2006, Chapter 3:Unit 3. 22.
[6]	Raposo G, Stoorvogel W. Extracellular vesicles:exosomes, microvesicles, and friends[J]. J Cell Biol, 2013, 200(4):373-383. doi: 10.1083/jcb.201211138 pmid: 23420871
[7]	Hata T, Murakami K, Nakatani H, et al. Isolation of bovine milk-derived microvesicles carrying mRNAs and microRNAs[J]. Biochem Biophys Res Commun, 2010, 396(2):528-533. doi: 10.1016/j.bbrc.2010.04.135 URL
[8]	Lakkaraju A, Rodriguez-Boulan E. Itinerant exosomes:emerging roles in cell and tissue polarity[J]. Trends Cell Biol, 2008, 18(5):199-209. doi: 10.1016/j.tcb.2008.03.002 pmid: 18396047
[9]	Rani P, Vashisht M, Golla N, et al. Milk miRNAs encapsulated in exosomes are stable to human digestion and permeable to intestinal barrier in vitro[J]. J Funct Foods, 2017, 34:431-439. doi: 10.1016/j.jff.2017.05.009 URL
[10]	Lonnerdal B, Du XG, Liao YL, et al. Human milk exosomes resist digestion in vitro and are internalized by human intestinal cells[J]. FASEB J, 2015, 29(S1):1213.
[11]	Chen T, Xie MY, Sun JJ, et al. Porcine milk-derived exosomes promote proliferation of intestinal epithelial cells[J]. Sci Rep, 2016, 6:33862. doi: 10.1038/srep33862 URL
[12]	Hock A, Miyake H, Li B, et al. Breast milk-derived exosomes promote intestinal epithelial cell growth[J]. J Pediatr Surg, 2017, 52(5):755-759. doi: 10.1016/j.jpedsurg.2017.01.032 URL
[13]	Zhang QZ, Ke HZ, Blikslager A, et al. Type III interferon restriction by porcine epidemic diarrhea virus and the role of viral protein nsp1 in IRF₁ signaling[J]. J Virol, 2018, 92(4):e01677-17.
[14]	Shen Z, Wang G, Yang YL, et al. A conserved region of nonstructural protein 1 from alphacoronaviruses inhibits host gene expression and is critical for viral virulence[J]. J Biol Chem, 2019, 294(37):13606-13618. doi: 10.1074/jbc.RA119.009713 URL
[15]	Zhang QZ, Ma JY, Yoo D. Inhibition of NF-κB activity by the porcine epidemic diarrhea virus nonstructural protein 1 for innate immune evasion[J]. Virology, 2017, 510:111-126. doi: 10.1016/j.virol.2017.07.009 URL
[16]	成温玉, 白云, 贾怀杰, 等. 猪流行性腹泻病毒蛋白拮抗宿主天然免疫应答的研究进展[J]. 生物技术通报, 2020, 36(12):229-238. doi: 10.13560/j.cnki.biotech.bull.1985.2020-0436
	Cheng WY, Bai Y, Jia HJ, et al. Research progress on proteins of PEDV antagonizing host innate immune responses[J]. Biotechnol Bull, 2020, 36(12):229-238.
[17]	曹丽艳. 猪流行性腹泻病毒感染猪小肠上皮细胞抑制IFN-β产生及激活NF-κB机理研究[D]. 哈尔滨:东北农业大学, 2015.
	Cao LY. The mechanism by which porcine epidemic diarrhea virus inhibits interferon-β production and activates NF-κB in porcine intestinal epithelial cells[D]. Harbin:Northeast Agricultural University, 2015.
[18]	Li F. Structure, function, and evolution of coronavirus spike proteins[J]. Annu Rev Virol, 2016, 3(1):237-261. doi: 10.1146/virology.2016.3.issue-1 URL
[19]	Yang LJ, Xu JY, Guo LJ, et al. Porcine epidemic diarrhea virus-induced epidermal growth factor receptor activation impairs the antiviral activity of type I interferon[J]. J Virol, 2018, 92(8):e02095-17.
[20]	Chen YF, Zhang ZB, Li J, et al. Porcine epidemic diarrhea virus S1 protein is the critical inducer of apoptosis[J]. Virol J, 2018, 15(1):170. doi: 10.1186/s12985-018-1078-4 URL
[21]	Samuel CE. Antiviral actions of interferons[J]. Clin Microbiol Rev, 2001, 14(4):778-809, table of contents. pmid: 11585785
[22]	Ding Z, Fang L, Jing H, et al. Porcine epidemic diarrhea virus nucleocapsid protein antagonizes beta interferon production by sequestering the interaction between IRF₃ and TBK₁[J]. J Virol, 2014, 88(16):8936-8945. doi: 10.1128/JVI.00700-14 URL
[23]	Guo LJ, Luo XL, Li R, et al. Porcine epidemic diarrhea virus infection inhibits interferon signaling by targeted degradation of STAT1[J]. J Virol, 2016, 90(18):8281-8292. doi: 10.1128/JVI.01091-16 URL
[24]	Sun M, Ma J, Yu Z, et al. Identification of two mutation sites in spike and envelope proteins mediating optimal cellular infection of porcine epidemic diarrhea virus from different pathways[J]. Vet Res, 2017, 48(1):44. doi: 10.1186/s13567-017-0449-y URL
[25]	Xu XG, Zhang HL, Zhang Q, et al. Porcine epidemic diarrhea virus E protein causes endoplasmic Reticulum stress and up-regulates interleukin-8 expression[J]. Virol J, 2013, 10:26. doi: 10.1186/1743-422X-10-26 URL
[26]	Xu XG, Zhang HL, Zhang Q, et al. Porcine epidemic diarrhea virus M protein blocks cell cycle progression at S-phase and its subcellular localization in the porcine intestinal epithelial cells[J]. Acta Virol, 2015, 59(3):265-275. pmid: 26435150
[27]	Kaewborisuth C, He QG, Jongkaewwattana A. The accessory protein ORF₃ contributes to porcine epidemic diarrhea virus replication by direct binding to the spike protein[J]. Viruses, 2018, 10(8):399. doi: 10.3390/v10080399 URL
[28]	Ye S, Li Z, Chen F, et al. Porcine epidemic diarrhea virus ORF₃ gene prolongs S-phase, facilitates formation of vesicles and promotes the proliferation of attenuated PEDV[J]. Virus Genes, 2015, 51(3):385-392. doi: 10.1007/s11262-015-1257-y URL
[29]	Si FS, Hu XX, Wang CY, et al. Porcine Epidemic Diarrhea Virus(PEDV)ORF3 Enhances Viral Proliferation by Inhibiting Apoptosis of Infected Cells[J]. Viruses, 2020, 12(2):214. doi: 10.3390/v12020214 URL
[30]	Meng F, Suo S, Zarlenga DS, et al. A phage-displayed peptide recognizing porcine aminopeptidase N is a potent small molecule inhibitor of PEDV entry[J]. Virology, 2014, 456/457:20-27. doi: 10.1016/j.virol.2014.01.010 URL
[31]	Shan Z, Yin J, Wang Z, et al. Identification of the functional domain of the porcine epidemic diarrhoea virus receptor[J]. J Gen Virol, 2015, 96(9):2656-2660. doi: 10.1099/vir.0.000211 URL
[32]	Park JE, Park ES, Yu JE, et al. Development of transgenic mouse model expressing porcine aminopeptidase N and its susceptibility to porcine epidemic diarrhea virus[J]. Virus Res, 2015, 197:108-115. doi: 10.1016/j.virusres.2014.12.024 URL
[33]	Wu J, Gao F, Xu T, et al. CLDN₁ induces autophagy to promote proliferation and metastasis of esophageal squamous carcinoma through AMPK/STAT1/ULK1 signaling[J]. J Cell Physiol, 2020, 235(3):2245-2259. doi: 10.1002/jcp.v235.3 URL
[34]	Mahati S, Xiao L, Yang Y, et al. miR-29a suppresses growth and migration of hepatocellular carcinoma by regulating CLDN₁[J]. Biochem Biophys Res Commun, 2017, 486(3):732-737. doi: 10.1016/j.bbrc.2017.03.110 URL
[35]	Pope JL, Ahmad R, Bhat AA, et al. Claudin-1 overexpression in intestinal epithelial cells enhances susceptibility to adenamatous polyposis coli-mediated colon tumorigenesis[J]. Mol Cancer, 2014, 13:167. doi: 10.1186/1476-4598-13-167 URL
[36]	Eftang LL, Esbensen Y, Tannæs TM, et al. Up-regulation of CLDN₁ in gastric cancer is correlated with reduced survival[J]. BMC Cancer, 2013, 13:586. doi: 10.1186/1471-2407-13-586 pmid: 24321518