猪Beclin1基因调控伪狂犬病病毒诱导细胞自噬和凋亡的机制分析

doi:10.11843/j.issn.0366-6964.2018.06.020

畜牧兽医学报 ›› 2018, Vol. 49 ›› Issue (6): 1274-1281.doi: 10.11843/j.issn.0366-6964.2018.06.020

猪Beclin1基因调控伪狂犬病病毒诱导细胞自噬和凋亡的机制分析

胡林¹, 南良康¹, 张敏¹, 许苏童¹, 杨了寒¹, 李帅峰¹, 王凯¹, 刘正飞², 张淑君^1*

1. 华中农业大学动物科学技术学院, 农业动物遗传育种与繁殖教育部重点实验室, 武汉 430070;
2. 华中农业大学动物医学院农业微生物学国家重点实验室, 武汉 430070

收稿日期:2017-09-14 出版日期:2018-06-23 发布日期:2018-06-22
通讯作者: 张淑君,教授,E-mail:sjxiaozhang@mail.hzau.edu.cn
作者简介:胡林(1991-),男,湖北武汉人,硕士,主要从事动物抗病遗传育种研究,E-mail:hulin877314193@163.com
基金资助:
国家自然科学基金（31572367；31272427）

The Mechanism of Porcine Beclin1 Gene on Autophagy and Apoptosis

HU Lin¹, NAN Liang-kang¹, ZHANG Min¹, XU Su-tong¹, YANG Liao-han¹, LI Shuai-feng¹, WANG Kai¹, LIU Zheng-fei², ZHANG Shu-jun^1*

1. Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction, Ministry of Education, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan 430070, China;
2. The State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China

Received:2017-09-14 Online:2018-06-23 Published:2018-06-22

摘要/Abstract

摘要：

旨在研究伪狂犬病病毒（PRV）感染后细胞自噬和凋亡情况，进一步分析鉴定PRV感染条件下与Beclin1互作的猪宿主凋亡家族蛋白。利用免疫印迹（WB）方法检测PRV感染细胞的自噬水平；利用流式细胞术探讨凋亡水平；最后利用免疫共沉淀试验分析PRV感染PK-15细胞中Beclin1与Bcl-2和Bcl-xL的结合水平。结果显示，PRV感染导致LC3-Ⅱ的表达明显增多，而P62显著下降，且AKT（S473）磷酸化水平显著减少；其次，PRV感染诱导细胞凋亡且显著上调剪切型Caspase-9/8的表达水平；最后，免疫共沉淀结果显示PRV感染条件下Beclin1能与Bcl-2和Bcl-xL结合，但结合的水平明显降低。PRV感染诱导的细胞自噬可能依赖于PI3K-Ⅰ/AKT信号通路，PRV感染可能同时激活了线粒体凋亡途经和死亡受体途经从而诱导细胞凋亡，而PRV感染诱导的细胞自噬和细胞凋亡可能通过抑制Beclin1和Bcl-2、Bcl-xL结合而相互关联。

关键词: PRV, 细胞自噬, 细胞凋亡, Beclin1

Abstract:

To study the relationship of autophagy and apoptosis after PK-15 cells were infected by pseudorabies virus (PRV), porcine host anti-apoptotic family proteins (Bcl-2 and Bcl-xL) interacted with Beclin1 were further analyzed. Western blotting (WB) was taken to measure the expression of LC3 and P62 proteins; and the level of apoptosis in PK-15 cells infected by PRV was investigated by Flow cytometry. Finally, Co-immunoprecipitation (Co-IP) was used to investigate the interaction between Beclin1 and Bcl-2/Bcl-xL. The expression of LC3-Ⅱ significantly increased, while that of P62 and p-AKT (S473) significantly decreased. In addition, the results of Flow cytometry showed that apoptosis was induced by PRV. Meanwhile, cleaved Caspase-9/8 significantly increased compared to control group detected by WB. Moreover, Co-IP results showed that the infection of PRV prevented the association between Beclin1/Bcl-2 and Beclin1/Bcl-xL. PRV induces autophagy which may depend on PI3K-I/AKT and apoptosis which simultaneously active Caspase-9 and Caspase-8, respectively. Autophagy might be induced through the interactions of Beclin1/Bcl-2 or Beclin1/Bcl-xL.

Key words: pseudorabies virus, autophagy, apoptosis, Beclin1

中图分类号:

胡林, 南良康, 张敏, 许苏童, 杨了寒, 李帅峰, 王凯, 刘正飞, 张淑君. 猪Beclin1基因调控伪狂犬病病毒诱导细胞自噬和凋亡的机制分析[J]. 畜牧兽医学报, 2018, 49(6): 1274-1281.

HU Lin, NAN Liang-kang, ZHANG Min, XU Su-tong, YANG Liao-han, LI Shuai-feng, WANG Kai, LIU Zheng-fei, ZHANG Shu-jun. The Mechanism of Porcine Beclin1 Gene on Autophagy and Apoptosis[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(6): 1274-1281.

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猪Beclin1基因调控伪狂犬病病毒诱导细胞自噬和凋亡的机制分析

The Mechanism of Porcine Beclin1 Gene on Autophagy and Apoptosis

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