猪肺炎支原体不同毒力菌株对猪气管上皮3D细胞的损伤差异与机制分析

doi:10.11843/j.issn.0366-6964.2018.02.017

摘要/Abstract

摘要：

为更真实地探究猪肺炎支原体（Mhp）不同毒力菌株对猪气管上皮细胞（STEC）的致病性差异与机制，通过气液界面培养技术（ALI），建立了传代STEC细胞系3D分化培养模型及Mhp在此细胞上的感染模型。将相同剂量的Mhp强毒株NJ株、中等毒力菌株AH株和弱毒株168L株分别感染3D培养的猪气管上皮细胞，分别通过单层细胞跨膜电阻检测、Alamar Blue检测、乳酸脱氢酶（LDH）释放检测等试验测定细胞单层完整性与细胞活性，并通过激光共聚焦和荧光分光光度计测定感染后48 h的细胞MUC5B黏液蛋白的分泌量，从细胞生长特性、活性和黏液分泌功能方面比较不同毒力Mhp菌株感染对STEC分化细胞的影响。研究还从细胞氧化系统方面，比较检测各感染组细胞内NO和内源性ROS分泌量，进一步探索Mhp的致病机制。结果表明：强毒株NJ株和中等毒力AH株组纤毛有明显的变粗、破损和脱落现象，而弱毒株168L株组对纤毛的影响较小；Mhp菌株感染后细胞单层跨膜电阻显著下降，且电阻下降程度与菌株毒力呈正相关。Alamar Blue检测试验、乳酸脱氢酶（LDH）释放检测结果均表明，Mhp NJ株与AH株感染后均可以显著降低细胞活性，且在感染后48 h最明显。Mhp菌株感染后还可促进分化细胞分泌MUC5B黏蛋白，菌株毒力越强，刺激黏蛋白分泌的量也越多。氧化应激检测结果显示，除Mhp弱毒菌株168L株外，中等毒力菌株AH株和强毒株NJ株感染3D分化的STEC细胞后，可显著刺激细胞分泌NO和内源性ROS，显著引起细胞的氧化应激反应。而经NAC抗氧化处理后，强毒株NJ株、中等毒力AH株和弱毒株168L株组细胞ROS和黏液分泌量均显著降低，细胞活性和电阻值均显著升高。本研究通过更接近体内环境的3D分化细胞感染模型证明，Mhp感染可对宿主细胞的生长特性与活性产生损伤，且损伤的程度与毒力呈正相关；而这种损伤机制与Mhp菌株引起感染细胞氧化应激的能力密切相关。

Abstract:

To study the pathogenic mechanism of Mycoplasma hyopneumoniae (Mhp), an air-liquid interface (ALI) cultivation model of continuous STEC cell line, closer to the status in vivo, was developed for Mhp infection. Mhp strains with different virulence (virulence strains NJ, intermediate virulence strain AH, and attenuated strain 168L) were selected for respective infection with the same dose. The integrity of cell monolayer and the vitality of the infected cells were respectively evaluated by TEER value, Alamar Blue assay and lactate dehydrogenase (LDH) release assay. The ability of the differentiated cells to secrete MUC5B mucin against Mhp infection was measured by laser confocal and fluorescence spectrophotometer. To further study the pathogenic mechanism of Mhp infection, the cellular oxidation system of the infected STEC cells, including the secretion of NO and endogenous ROS were detected, respectively. The effect of NAC antioxidant on cell damage of Mycoplasma infection group was observed and was further demonstrated. The results revealed that the infection of virulence strains NJ and intermediate virulence strains AH could significantly roughen, disorder or damage the microvillus and cilia on the surface of STEC cells, but not attenuated strains 168L. The resistance of cell monolayer was decreased after infection, which was positively correlated with the virulence of Mhp strains. Mhp strains NJ and AH could significantly reduce the vitality of the infected cells using Alamar Blue and LDH release assays, especially at 48 h post-infection. Mhp strains could also promote the differentiated cells to secrete MUC5B mucus against Mhp infection, and the stronger the virulence of the strain was, the more mucous was secreted. It was found that except the 168L strain, AH strain and NJ strain could both significantly stimulate STEC cells to secrete NO and endogenous ROS, which could cause the oxidative stress reaction of cells significantly. After the anti-oxidation treatment of NAC, the ROS and mucus secretion of virulence strains NJ, intermediate virulence strains AH and the attenuated strains 168L groups decreased significantly, while the cell activity and resistance value increased significantly, while control group were not significantly changed. This study has been proved that Mhp infection can comprehensively disorder the growth characteristics, vitality and secretory function of the host cells, which are positively correlated with the virulence of Mhp strains. Moreover, the pathogenic mechanism of Mhp infection is closely related to the ability to induce the oxidative stress to host cells.

中图分类号:

S852.62

冯艳艳, 王海燕, 刘蓓蓓, 韦艳娜, 张珍珍, 白昀, 倪博, 邵国青, 雷治海, 冯志新. 猪肺炎支原体不同毒力菌株对猪气管上皮3D细胞的损伤差异与机制分析[J]. 畜牧兽医学报, 2018, 49(2): 378-387.

FENG Yan-yan, WANG Hai-yan, LIU Bei-bei, WEI Yan-na, ZHANG Zhen-zhen, BAI Yun, NI Bo, SHAO Guo-qing, LEI Zhi-hai, FENG Zhi-xin. The Pathogenic Mechanism of Mycoplasma Hyopneumoniae Strains with Different Virulence on 3D Cultured Swine Tracheal Epithelial Cells[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(2): 378-387.

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