山羊副流感病毒3型通过Caspase途径诱导气管上皮细胞凋亡

doi:10.11843/j.issn.0366-6964.2019.10.012

畜牧兽医学报 ›› 2019, Vol. 50 ›› Issue (10): 2061-2069.doi: 10.11843/j.issn.0366-6964.2019.10.012

山羊副流感病毒3型通过Caspase途径诱导气管上皮细胞凋亡

廖政^1,3, 李基棕^1,2*, 肖芳^1,3, 钟纯燕^1,3, 杨蕾蕾¹, 毛立¹, 李文良¹, 孙敏¹, 主性³, 嵇辛勤³, 张纹纹¹, 刘茂军¹

1. 江苏省农业科学院兽医研究所, 兽医诊断检测重点实验室, 农业部兽用生物制品工程技术重点实验室, 南京 210014;
2. 临沂大学药学院, 临沂 276000;
3. 贵州大学动物科学学院, 贵阳 550025

收稿日期:2019-04-10 出版日期:2019-10-23 发布日期:2019-10-23
通讯作者: 李基棕,主要从事动物传染病防治和诊断技术研究,Tel:025-84391152,E-mail:lijizong22@sina.com
作者简介:廖政(1993-),男,贵州黄平人,硕士生,主要从事动物传染病防治和诊断技术研究,Tel:025-84391152,E-mail:320414112@qq.com
基金资助:
国家自然科学基金（31702272；31802196）；江苏省自然科学基金（BK2017059）

Caprine Parainfluenza Virus Type 3 Induces Apoptosis in Goat Bronchial Epithelial Cells via Caspase-mediated Pathways

LIAO Zheng^1,3, LI Jizong^1,2*, XIAO Fang^1,3, ZHONG Chunyan^1,3, YANG Leilei¹, MAO Li¹, LI Wenliang¹, SUN Min¹, ZHU Xing³, JI Xinqin³, ZHANG Wenwen¹, LIU Maojun¹

1. Key Laboratory of Veterinary Diagnosis, Key Laboratory of Veterinary Biological Engineering and Technology of Ministry of Agriculture, Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China;
2. School of Pharmacy, Linyi University, Linyi 276000, China;
3. College of Animal Science of Guizhou University, Guiyang 550025, China

Received:2019-04-10 Online:2019-10-23 Published:2019-10-23

摘要/Abstract

摘要： 旨在揭示山羊副流感病毒3型（CPIV3）感染气管上皮细胞后是否可诱导细胞发生凋亡，并对细胞凋亡的信号通路进行初步探究。本研究将CPIV3病毒液接种气管上皮细胞，在12、24、36、48、72和96 h收集培养物上清检测病毒增殖滴度；通过形态学观察CPIV3诱导气管上皮细胞病变（CPE）情况；采用Annexin V-FITC/PI双染细胞凋亡检测试剂盒和Caspase-3、Caspase-8和Caspase-9活性检测试剂盒检测凋亡水平及相关指标；荧光定量PCR检测细胞凋亡分子mRNA表达水平；Western blot分析激活型Caspase-3蛋白表达变化情况。结果显示，CPIV3在气管上皮细胞中的增殖呈上升趋势，96 h能达到10^4.50TCID₅₀·mL^-1；形态学观察发现，病毒接种后48 h出现细胞收缩变圆、脱落等CPE现象；流式细胞术检测及Caspase活性检测表明，感染组细胞出现细胞凋亡，48 h后细胞凋亡率达19.66%，且Caspase-3、Caspase-8和Caspase-9活性随着时间延长逐渐升高；死亡受体凋亡途径和线粒体凋亡途径细胞凋亡因子mRNA表达上调。Western blot分析揭示，激活型Caspase-3蛋白在病毒感染过程中被活化。本研究证实CPIV3感染可诱导气管上皮细胞凋亡，且Caspase途径在病毒诱导细胞凋亡的过程中发挥重要作用。

Abstract: To investigate the molecular mechanisms of apoptosis in goat bronchial epithelial cells infected with CPIV3 in vitro, the cells were inoculated with CPIV3 JS2013 strain that isolated from the previous study. The cultural supernatants of the cells were harvested at 12,24,36,48,72 and 96 h post inoculation(hpi), then qRT-PCR and TCID₅₀ assays were conducted to determine CPIV3 proliferation levels. The apoptosis was examined by Annein V-FITC/PI double staining cell apoptosis detection kit; and relevant indicators were detected by Caspase-3, Caspase-8 and Caspase-9 activity detection kit, qRT-PCR assay, and Western blot. The results showed that virus titers increased from 10^1.80 to 10^4.50 TCID₅₀·mL^-1 at 96 hpi. Observation with inverted microscope showed that CPIV3-infected cells had typical CPE, including shrink and disrupted. Flow cytometry analysis of apoptosis showed that the number of apoptotic cells increased to 19.66% at 48 hpi. Caspases activity assay demonstrated that CPIV3 infection increased the activation of Caspase-3, Caspase-8 and Caspase-9. Meanwhile, the expression levels of intrinsic pathway and extrinsic apoptotic pathway mRNA were increased. In addition, cleaved Caspase-3 was activated in CPIV3-infected cells. These findings indicate that CPIV3 infection could induce apoptosis and the activation of Caspase played important role in apoptosis.

中图分类号:

廖政, 李基棕, 肖芳, 钟纯燕, 杨蕾蕾, 毛立, 李文良, 孙敏, 主性, 嵇辛勤, 张纹纹, 刘茂军. 山羊副流感病毒3型通过Caspase途径诱导气管上皮细胞凋亡[J]. 畜牧兽医学报, 2019, 50(10): 2061-2069.

LIAO Zheng, LI Jizong, XIAO Fang, ZHONG Chunyan, YANG Leilei, MAO Li, LI Wenliang, SUN Min, ZHU Xing, JI Xinqin, ZHANG Wenwen, LIU Maojun. Caprine Parainfluenza Virus Type 3 Induces Apoptosis in Goat Bronchial Epithelial Cells via Caspase-mediated Pathways[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(10): 2061-2069.

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山羊副流感病毒3型通过Caspase途径诱导气管上皮细胞凋亡

Caprine Parainfluenza Virus Type 3 Induces Apoptosis in Goat Bronchial Epithelial Cells via Caspase-mediated Pathways

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