SLT-Ⅱe对PIMVECs分泌炎症反应相关细胞因子的影响

doi:10.16431/j.cnki.1671-7236.2020.11.007

摘要/Abstract

摘要： 本试验旨在探究大肠杆菌志贺样毒素Ⅱ型变异体（SLT-Ⅱe）处理后，猪小肠微血管内皮细胞（PIMVECs）分泌的肿瘤坏死因子α（TNF-ɑ）、P选择素（P-selectin）、一氧化氮（NO）、细胞间黏附分子1（ICAM-1）和内皮素（ET）水平的变化。采用超速离心法提取SLT-Ⅱe，采用BCA试剂盒测定其质量浓度，使用SDS-PAGE及Western blotting验证蛋白纯度；用WST-1法从0.1、0.5、1、5、10 μg/mL SLT-Ⅱe和0、2、4、8、12、24 h中筛选出SLT-Ⅱe对PIMVECs最佳作用浓度与时间；ELISA法检测对照组（0 μg/mL SLT-Ⅱe）和试验组（1 μg/mL SLT-Ⅱe）细胞上清中TNF-ɑ、P-selectin、NO、ICAM-1和ET含量的变化。结果表明：所提蛋白为SLT-Ⅱe，蛋白含量为721.75 μg/mL；1 μg/mL的SLT-Ⅱe作用8 h后可极显著降低PIMVECs的活性（P<0.01）；1 μg/mL的SLT-Ⅱe作用9和12 h时PIMVECs的TNF-ɑ分泌量显著升高（P<0.05），NO/ET的值显著高于对照组（P<0.05），作用6、9、12 h时P-selectin的含量显著升高（P<0.05），作用12 h时的ICAM-1分泌量显著升高（P<0.05）。综上所述，SLT-Ⅱe浓度为1 μg/mL时可以诱导PIMVECs分泌的TNF-ɑ、P-selectin、ICAM-1以及NO/ET上升，为研究仔猪水肿病发病机制提供参考。

关键词: 志贺样毒素Ⅱ型变异体; 小肠微血管内皮细胞; 炎症反应细胞因子; 猪<

Abstract: The aim of the study was to investigate the changes of TNF-α,P-selectin,ICAM-1,NO and ET in porcine intestinal microvascular endothelial cells (PIMVECs) treated with Escherichia coli Shiga-like toxin type Ⅱ variant (SLT-Ⅱe).SLT-Ⅱe was extracted from Escherichia coli by using ultracentrifugation,and the concentration was determined by BAC kit.The purity of SLT-Ⅱe was detected by SDS-PAGE and Western blotting.The optimal incubation time (0,2,4,8,12,24 h) and concentration of SLT-Ⅱe (0.1,0.5,1,5,10 μg/mL) on PIMVECs were evaluated by WST-1.The levels of TNF-α,P-selectin,ICAM-1,NO and ET in cell culture supernatant of control group (0 μg/mL SLT-Ⅱe) and test group (1 μg/mL SLT-Ⅱe) were detected by ELISA.The results showed that using ultracentrifugation could extract pure SLT-Ⅱe with a concentration of 721.75 μg/mL.1 μg/mL SLT-Ⅱe could significantly reduce the activity of PIMVECs after 8 h (P<0.01) and significantly increase the TNF-α secretion of PIMVECs after 9 and 12 h treatment (P<0.05).The NO/ET values at 9 and 12 h were significantly higher than that in the control group (P<0.05).After 9 and 12 h treatment the content of P-selectin was significantly increased (P<0.05) and the levels of ICAM-1 was significantly increased at 12 h (P<0.05).In summary,1 μg/mL SLT-Ⅱe could induce the increase of TNF-α,P-selectin,ICAM-1 and NO/ET secreted by PIMVECs,which laid a foundation for studying the pathogenesis of piglet edema disease.

Key words: Shiga-like toxin type Ⅱ variant; microvascular endothelial cells; inflammatory cytokines; pigs

中图分类号:

王伟然, 施晓杰, 张蒙, 穆祥. SLT-Ⅱe对PIMVECs分泌炎症反应相关细胞因子的影响[J]. 中国畜牧兽医, 2020, 47(11): 3467-3473.

WANG Weiran, SHI Xiaojie, ZHANG Meng, MU Xiang. Effects of SLT-Ⅱe on Cytokines of Inflammatory Response Secreted by PIMVECs[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(11): 3467-3473.

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