猪圆环病毒Ⅱ型体外诱导3D4/2细胞炎症模型的建立

doi:10.16431/j.cnki.1671-7236.2022.12.039

摘要/Abstract

摘要： 【目的】通过对猪圆环病毒Ⅱ型（PCV2）体外感染3D4/2细胞浓度、时间与细胞炎症水平进行探讨，建立PCV2体外感染3D4/2细胞炎症反应模型，以期为后期药物调控PCV2诱发3D4/2细胞炎症反应的研究奠定基础。【方法】将3D4/2细胞分为对照组及10⁰、10^-1、10^-2和10^-3 PCV2感染组，每组3个重复。对照组用DMEM培养，各PCV2感染组用不同稀释倍数PCV2液培养，2 h后均更换为含5%胎牛血清（FBS）的DMEM维持液进行培养，培养4、8、12和24 h后分别收集细胞及细胞上清液。采用Griess法检测一氧化氮（NO）水平，DCFH-DA荧光探针法检测活性氧（ROS）水平，酶标法检测还原型谷胱甘肽（GSH）水平，分光光度法检测黄嘌呤氧化酶（XOD）和髓过氧化物酶（MPO）活性，ELISA法测定白细胞介素-1β（IL-1β）、IL-6、肿瘤坏死因子-α（TNF-α）、IL-10、γ干扰素（IFN-γ）、IL-8、单核细胞趋化蛋白1（MCP-1）以及环氧合酶1（COX-1）和COX-2的分泌水平。【结果】10⁰至10^-3 PCV2作用4、8、12和24 h均能够成功感染3D4/2细胞。与对照组相比，10⁰ PCV2在感染3D4/2细胞4、8、12、24 h后ROS水平均极显著升高（P<0.01），10^-1至10^-3 PCV2感染3D4/2细胞8、12、24 h后ROS水平显著或极显著升高（P<0.05；P<0.01）；10⁰至10^-3 PCV2感染3D4/2细胞8、12、24 h后，细胞内NO浓度及MPO活性显著提高（P<0.05），细胞上清液中的IL-1β、IL-6、TNF-α、IL-10、IFN-γ、IL-8和MCP-1水平及COX-1活性均显著或极显著升高（P<0.05；P<0.01），其中10⁰ PCV2感染3D4/2细胞后，各炎症因子水平上升最显著，且随着时间的延长，NO浓度逐渐升高，XOD活性逐渐降低。【结论】PCV2可诱导3D4/2细胞炎症反应，且10⁰ PCV2体外感染3D4/2细胞4~12 h是建立炎症模型的最佳条件。

关键词: 猪圆环病毒Ⅱ型(PCV2); 炎性因子; 3D4/2细胞

Abstract: 【Objective】 The experiment was to establish a model of the inflammatory response of 3D4/2 cells infected with Porcine circovirus type 2 (PCV2) in vitro by exploring the relationship between the concentration, time and the inflammatory level of PCV2 in vitro infection, and lay a foundation for the study of the later drug regulation of PCV2-induced 3D4/2 cells inflammatory response.【Method】 3D4/2 cells were divided into control group and different dilutions PCV2 infected groups (10⁰, 10^-1, 10^-2 and 10^-3 dilutions), with 3 replicates per group.The control group was treated with DMEM, and PCV2 infected groups were treated with PCV2 in different concentrations, then replaced with DMEM maintenance solution containing 5% FBS for culture after 2 h.After 4, 8, 12 and 24 h of culture, cells and cell supernatant were collected, respectively.NO level was detected by Griess method, ROS level was detected by DCFH-DA fluorescent probe method, GSH level was detected by enzyme-conjugated method, XOD and MPO enzyme activities were detected by spectrophotometry.IL-1β, IL-6, TNF-α, IL-10, IFN-γ, IL-8, MCP-1, COX-1 and COX-2 inflammatory factor levels were determined by ELISA method.【Result】 10⁰ to 10^-3 concentrations of PCV2 could successfully infect 3D4/2 cells after 4, 8, 12 and 24 h.Compared with control group, after 3D4/2 cells were infected with PCV2 at 10⁰ concentrations for 4, 8, 12 and 24 h, the ROS level was extremely significantly increased (P<0.01), and after 3D4/2 cells were infected with PCV2 at 10^-1 to 10^-3 concentrations for 8, 12 and 24 h, the ROS level was significantly or extremely significantly increased (P<0.05;P<0.01).After 3D4/2 cells were infected with 10⁰ to 10^-3 concentrations of PCV2 for 8, 12 and 24 h, the intracellular NO concentration and MPO activity were significantly increased (P<0.05).The levels of IL-1β, IL-6, TNF-α, IL-10, IFN-γ, IL-8, MCP-1 and the activity of COX-1 in cell supernatant or cells were significantly or extremely significantly increased (P<0.05;P<0.01).Among which, after 10⁰ concentration of PCV2 infected 3D4/2 cells, the increase of each inflammatory factor was the most significant, and with the prolongation of time, the NO concentration was gradually increased, and the activity of XOD was gradually decreased.【Conclusion】 PCV2 could induce inflammatory responses in 3D4/2 cells, and infection of 3D4/2 cells with 10⁰ dilution PCV2 in vitro for 4-12 h was the best condition for establishing an inflammatory model.

Key words: Porcine circovirus type 2 (PCV2); inflammatory cytokines; 3D4/2 cells

中图分类号:

S858.28

赵怡, 张文, 陈家骥, 宋漫玲, 陈奇, 谢小东, 胡庭俊. 猪圆环病毒Ⅱ型体外诱导3D4/2细胞炎症模型的建立[J]. 中国畜牧兽医, 2022, 49(12): 4920-4927.

ZHAO Yi, ZHANG Wen, CHEN Jiaji, SONG Manling, CHEN Qi, XIE Xiaodong, HU Tingjun. Establishment of 3D4/2 Cell Inflammatory Model Induced by PCV2 in vitro[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(12): 4920-4927.

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