猪繁殖与呼吸综合征病毒-脂多糖刺激对中性粒细胞黏附猪肺微血管内皮细胞的影响

doi:10.16431/j.cnki.1671-7236.2022.02.008

摘要/Abstract

摘要： 【目的】研究猪繁殖与呼吸综合征病毒(PRRSV)单独和联合脂多糖(LPS)刺激对肺脏募集中性粒细胞(Neu)的影响, 以及猪肺微血管内皮细胞(MVECs)在其中的作用。【方法】取约10日龄仔猪的肺脏组织, 采用Ⅱ型胶原酶消化和差速贴壁法, 分离培养原代MVECs, 采用密度梯度离心法分离猪外周血Neu; 以PRRSV HN株或PRRSV-LPS刺激Neu和MVECs后, 将Neu加入培养MVECs的培养板, 孵育1 h, 4%多聚甲醛固定, 瑞氏染色, 观察并计数分析各组黏附的Neu数量; 采用免疫细胞化学染色法检测MVECs对P-选择素和E-选择素的表达; 采用虎红溶液染色法测定分析P-选择素和E-选择素抗体封闭MVECs时, PRRSV HN株或PRRSV-LPS刺激对Neu黏附于MVECs数量的影响。【结果】分离培养的MVECs呈CD34免疫荧光染色阳性, 阳性率约为92%;PRRSV HN株刺激18 h, 黏附于MVECs的Neu数量显著增加(P<0.05);PRRSV-LPS联合刺激时, 黏附的Neu数量显著多于LPS单独刺激(P<0.05);PRRSV或PRRSV-LPS刺激Neu和MVECs二者时, 黏附Neu数量的增加比单独刺激MVECs或Neu更明显; 免疫细胞化学染色显示, MVECs强阳性表达P-选择素和E-选择素; 用P-选择素和E-选择素抗体封闭MVECs时, PRRSV或PRRSV-LPS刺激诱导的Neu黏附增加呈不同程度下降, E-选择素封闭时具有显著差异(P<0.05)。【结论】 PRRSV感染能促进Neu与MVECs黏附, 亦能增加后继LPS刺激时黏附的Neu数量, MVECs表达的E-选择素是介导PRRSV致Neu黏附增加的重要分子之一。

关键词: 猪繁殖与呼吸综合征病毒(PRRSV); 中性粒细胞(Neu); 微血管内皮细胞(MVECs); 黏附; 选择素

Abstract: 【Objective】 This study was aimed to study the effects of Porcine reproductive and respiratory syndrome virus (PRRSV) alone and in combination with lipopolysaccharide (LPS) stimulation on lung recruitment neutrophil (Neu), and the role of porcine lung microvascular endothelial cells (MVECs).【Method】 The primary MVECs were isolated and cultured by type Ⅱ collagenase digestion and differential adhesion method.Neu was isolated from porcine peripheral blood by density gradient centrifugation.After stimulating Neu and MVECs with PRRSV HN strain or PRRSV-LPS, Neu was added to the culture plate with MVECs, incubated for 1 h, fixed with 4% paraformaldehyde, stained with Wright's staining, and the number of Neu adhered in each group was observed, counted and analyzed.The expression of P-selectin and E-selectin in MVECs was detected by immunocytochemical staining.The quantitative changes of Neu adhesion to MVECs stimulated by PRRSV HN strain or PRRSV-LPS were measured and analyzed by rose bengal solution staining when MVECs were blocked by P-selectin and E-selectin antibodies.【Result】 The isolated and cultured MVECs were CD34 immunofluorescence positive, and the positive rate was about 92%.When PRRSV HN strain was stimulated for 18 h, the number of Neu adhered to MVECs increased significantly (P < 0.05).When stimulated with PRRSV-LPS, the number of adherent Neu was significantly higher than that stimulated with LPS alone (P < 0.05).When PRRSV or PRRSV-LPS stimulated Neu and MVECs, the increase in the number of adherent Neu was more obvious than that only stimulated MVECs or Neu.Immunocytochemical staining showed that MVECs strongly expressed P-selectin and E-selectin.When P-selectin and E-selectin antibodies blocked MVECs, the increase of Neu adhesion induced by PRRSV or PRRSV-LPS decreased in varying degrees, and there was a significant difference when E-selectin blocked MVECs (P < 0.05).【Conclusion】 PRRSV infection could not only promote the adhesion between Neu and MVECs, but also increase the number of Neu adhered during subsequent LPS stimulation.E-selectin expressed by MVECs was one of the important molecules mediating the increase of Neu adhesion caused by PRRSV.

Key words: Porcine reproductive and respiratory syndrome virus (PRRSV); neutrophil (Neu); microvascular endothelial cells (MVECs); adhesion; selectin

中图分类号:

S852.65⁺1

王兆丽, 杨思宇, 吴艳梅, 宋晓晓, 穆祥, 张涛. 猪繁殖与呼吸综合征病毒-脂多糖刺激对中性粒细胞黏附猪肺微血管内皮细胞的影响[J]. 中国畜牧兽医, 2022, 49(2): 471-479.

WANG Zhaoli, YANG Siyu, WU Yanmei, SONG Xiaoxiao, MU Xiang, ZHANG Tao. Effects of PRRSV-LPS Treatment on Neutrophil Adhesion to Porcine Pulmonary Microvascular Endothelial Cells[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(2): 471-479.

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