捻转血矛线虫半胱氨酸蛋白酶对山羊PBMCs免疫功能的影响

doi:10.11843/j.issn.0366-6964.2018.04.018

畜牧兽医学报 ›› 2018, Vol. 49 ›› Issue (4): 804-810.doi: 10.11843/j.issn.0366-6964.2018.04.018

捻转血矛线虫半胱氨酸蛋白酶对山羊PBMCs免疫功能的影响

胡孟娟, 周丽娜, 牛延萍, 徐立新, 宋小凯, 李祥瑞, 严若峰*

南京农业大学动物医学院, 南京 210095

收稿日期:2017-09-14 出版日期:2018-04-23 发布日期:2018-04-23
通讯作者: 严若峰(1976-),博士,教授,主要从事兽医寄生虫分子与免疫研究,E-mail:yanruofeng@njau.edu.cn
作者简介:胡孟娟(1991-),女,硕士生,主要从事寄生虫分子与免疫研究,E-mail:2015107074@njau.edu.cn,Tel:025-84395904。
基金资助:
中央高校基本科研业务费（KYYJ201710）；国家重点基础发展研究计划（973计划）（2015CB150300）；江苏高校优势学科建设工程资助项目（PAPD）

Effects of Haemonchus contortus Cysteine Protease on Immune Functions of Goat PBMCs

HU Meng-juan, ZHOU Li-na, NIU Yan-ping, XU Li-xin, SONG Xiao-kai, LI Xiang-rui, YAN Ruo-feng*

College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China

Received:2017-09-14 Online:2018-04-23 Published:2018-04-23

摘要/Abstract

摘要：

旨在探究捻转血矛线虫半胱氨酸蛋白酶（Hc58）对山羊外周血单个核细胞（PBMCs）免疫功能的影响。采集健康山羊血液，分离出PBMCs和单核细胞，分别以质量浓度为0、10、20、40 μg·mL^-1的重组蛋白Hc58与细胞体外共培养，采用硝酸还原酶法测定不同浓度Hc58对单核细胞分泌一氧化氮（NO）的影响；采用流式细胞术检测重组蛋白对单核细胞吞噬FITC-dextran能力的影响；采用荧光定量PCR检测PBMCs中IL-2、IL-4、IL-10、IL-17、IFN-γ和TGF-β的mRNA转录情况；采用迁移小室研究重组蛋白对PBMCs迁移的影响；采用流式细胞术检测不同浓度Hc58对PBMCs凋亡的影响。研究表明重组蛋白Hc58显著促进山羊单核细胞分泌NO；并且增强了山羊单核细胞的吞噬能力；Hc58显著上调IL-2、IL-4、IL-17、IFN-γ的表达量；Hc58显著提高山羊PBMCs凋亡比例，也使PBMCs迁移率增加。捻转血矛线虫半胱氨酸蛋白酶能通过多种途径影响宿主PBMCs发挥免疫作用。

Abstract:

The present study was designed to investigate the effects of Haemonchus contortus Cysteine protease on immune functions of peripheral blood mononuclear cells (PBMCs) of goat. PBMCs were separated from goat blood sample, monocytes were isolated from PBMCs because of their adherent growth. Recombinant protein of different concentrations (0, 10, 20, 40 μg·mL^-1) was cultured with PBMCs or monocytes in vitro. Nitric oxide (NO) secretion was measured by nitrate reductase assay. Cell migration was tested by Cell Culture Inserts. The mRNA transcriptional levels of IL-2, IL-4, IL-10, IL-17, IFN-γ and TGF-β were detected by real-time fluorescent quantitative PCR. Then cell phagocytosis and apoptosis were calculated by flow cytometry. The result revealed that Hc58 was able to dramatically enhance NO release and phagocytosis of monocytes. Hc58 might significantly up-regulate the production of IL-2, IL-4, IL-17 and IFN-γ. Furthermore, it also could notably increase apoptosis ratio and migration of PBMCs. There were signs that Haemonchus contortus Cysteine protease impacted on the immune functions of goat PBMCs via different pathways.

中图分类号:

S852.73

胡孟娟, 周丽娜, 牛延萍, 徐立新, 宋小凯, 李祥瑞, 严若峰. 捻转血矛线虫半胱氨酸蛋白酶对山羊PBMCs免疫功能的影响[J]. 畜牧兽医学报, 2018, 49(4): 804-810.

HU Meng-juan, ZHOU Li-na, NIU Yan-ping, XU Li-xin, SONG Xiao-kai, LI Xiang-rui, YAN Ruo-feng. Effects of Haemonchus contortus Cysteine Protease on Immune Functions of Goat PBMCs[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(4): 804-810.

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捻转血矛线虫半胱氨酸蛋白酶对山羊PBMCs免疫功能的影响

Effects of Haemonchus contortus Cysteine Protease on Immune Functions of Goat PBMCs

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