TGF-β1信号通路在副猪嗜血杆菌侵入3D4/21细胞中的作用

doi:10.11843/j.issn.0366-6964.2018.08.017

畜牧兽医学报 ›› 2018, Vol. 49 ›› Issue (8): 1720-1726.doi: 10.11843/j.issn.0366-6964.2018.08.017

TGF-β1信号通路在副猪嗜血杆菌侵入3D4/21细胞中的作用

沈懿娟, 周妮妮, 张健淞, 王美芬, 李玉峰*, 姜平

南京农业大学动物医学院农业部细菌学重点开放实验室, 南京 210095

收稿日期:2018-01-16 出版日期:2018-08-23 发布日期:2018-08-23
通讯作者: 李玉峰,E-mail:yufengli@njau.edu.cn
作者简介:沈懿娟(1991-),江苏苏州人,硕士,主要从事副猪嗜血杆菌致病机制的研究,E-mail:2015107059@njau.edu.cn
基金资助:
国家自然科学基金（31672565）；国家重点研究发展计划（2016YFD0500701-3）；江苏省高校重点学科建设项目（PAPD）

TGF-β1 Signaling Stimulates Invasion of Haemophilus parasuis into 3D4/21 Cells

SHEN Yi-juan, ZHOU Ni-ni, ZHANG Jian-song, WANG Mei-fen, LI Yu-feng*, JIANG Ping

Key Laboratory of Animal Bacteriology of Ministry of Agriculture, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China

Received:2018-01-16 Online:2018-08-23 Published:2018-08-23

摘要/Abstract

摘要：

本研究旨在探讨猪肺泡巨噬细胞传代细胞系3D4/21中转化生长因子β1（TGF-β1）表达和纤连蛋白（Fn）/α5整合素与副猪嗜血杆菌侵入的关系。通过吸附/侵入试验选择副猪嗜血杆菌最佳吸附/侵入条件；运用荧光定量PCR和ELISA方法检测副猪嗜血杆菌感染细胞后对TGF-β1表达的影响；用TGF-β1预处理3D4/21细胞后，用CFU检测TGF-β1表达对副猪嗜血杆菌侵入的影响；用流式细胞术检测副猪嗜血杆菌感染细胞后Fn/α5整合素表达的变化；用特异的小干扰RNA（siRNA）片段干扰Fn/α5整合素表达水平后，研究其与副猪嗜血杆菌对3D4/21吸附/侵入的影响。结果显示，副猪嗜血杆菌感染细胞后会导致TGF-β1表达增加；用重组TGF-β1预处理3D4/21细胞可以显著提高副猪嗜血杆菌的侵入水平；流式细胞术检测显示，重组TGF-β1和副猪嗜血杆菌分别处理3D4/21细胞可以增加Fn和α5整合素的表达；siRNA干扰3D4/21细胞中Fn和α5整合素的表达，细菌侵入数显著降低；然而，只有当α5整合素被干扰时，副猪嗜血杆菌的吸附数量下降。这些结果表明，TGF-β1和Fn/α5整合素的表达促进了副猪嗜血杆菌对3D4/21细胞的侵入，并在此过程中发挥重要作用。

Abstract:

This study aimed to investigate the relationship between the expression of transforming growth factor β1 (TGF-β1), leading to the expression of fibronectin (Fn)/α5 integrin, and the invasion of Haemophilus parasuis (H. parasuis) in porcine alveolar macrophage cell line 3D4/21. The optimal adherence/invasion conditions of H. parasuis to 3D4/21 cells were determined by a series of adherence/invasion experiments. TGF-β1 expression level in the cells infected by H. parasuis was detected using Q-PCR and ELISA. Treatment of cells with recombinant TGF-β1 prior to H. parasuis infection, CFU and flow cytometry were used to confirm the relationship between bacteria variation and the expression of TGF-β1. siRNA interference was used to down regulate the expression of Fn/α5 to confirm the relationship between the expression of Fn/α5 integrin and bacteria invasion. The H. parasuis infection resulted in the elevation of TGF-β1 expression in cells. Treatment of 3D4/21 cells with TGF-β1 prior to infection significantly increased the invasion of H. parasuis. Flow cytometry revealed that the expression of Fn/α5 integrin was increased in 3D4/21 cells treated with recombinant TGF-β1 and H. parasuis. Small interfering RNAs (siRNA) were used to down regulate the expression of Fn and α5 integrin in 3D4/21 cells, which significantly decreased the invasion of H. parasuis. However, H. parasuis attachment was decreased only on cells treated with the α5 integrin siRNA, there was no corresponding decrease in attachment to cells treated with Fn siRNA. These results demonstrate that expression of TGF-β1 and α5 integrin promote the invasion of H. parasuis to 3D4/21 cells and play an important role during this process.

中图分类号:

S852.613

沈懿娟, 周妮妮, 张健淞, 王美芬, 李玉峰, 姜平. TGF-β1信号通路在副猪嗜血杆菌侵入3D4/21细胞中的作用[J]. 畜牧兽医学报, 2018, 49(8): 1720-1726.

SHEN Yi-juan, ZHOU Ni-ni, ZHANG Jian-song, WANG Mei-fen, LI Yu-feng, JIANG Ping. TGF-β1 Signaling Stimulates Invasion of Haemophilus parasuis into 3D4/21 Cells[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(8): 1720-1726.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.xmsyxb.com/CN/10.11843/j.issn.0366-6964.2018.08.017

https://www.xmsyxb.com/CN/Y2018/V49/I8/1720

参考文献

[1] MØLLER K, ANDERSEN L V, CHRISTENSEN G, et al. Optimalization of the detection of NAD dependent Pasteurellaceae from the respiratory tract of slaughterhouse pigs[J]. Vet Microbiol, 1993, 36(3-4):261-271.
[2] KIELSTEIN P, RAPP-GABRIELSON V J. Designation of 15 serovars of Haemophilus parasuis on the basis of immunodiffusion using heat-stable antigen extracts[J]. J Clin Microbiol, 1992, 30(4):862-865.
[3] RÚBIES X, KIELSTEIN P, COSTA L, et al. Prevalence of Haemophilus parasuis serovars isolated in Spain from 1993 to 1997[J]. Vet Microbiol, 1999, 66(3):245-248.
[4] FRANDOLOSO R, PIVATO M, MARTÍNEZ-MARTÍNEZ S, et al. Differences in Haemophilus parasuis adherence to and invasion of AOC-45 porcine aorta endothelial cells[J]. BMC Vet Res, 2013, 9:207.
[5] WANG Y, LIU C, FANG Y, et al. Transcription analysis on response of porcine alveolar macrophages to Haemophilus parasuis[J]. BMC Genomics, 2012, 13:68.
[6] KING P T, SHARMA R. The lung immune response to Nontypeable Haemophilus influenzae (Lung Immunity to NTHi)[J]. J Immunol Res, 2015, 2015:706376.
[7] BELLO-ORTÍB, HOWELL K J, TUCKER A W, et al. Metatranscriptomics reveals metabolic adaptation and induction of virulence factors by Haemophilus parasuis during lung infection[J]. Vet Res, 2015, 46:102.
[8] AMANO H, SHIBATA M, KAJIO N, et al. Pathologic observations of pigs intranasally inoculated with serovar 1, 4 and 5 of Haemophilus parasuis using immunoperoxidase method[J]. J Vet Med Sci, 1994, 56(4):639-644.
[9] 汪伟, 王小敏, 何孔旺, 等. 猪圆环病毒2型体外刺激3D4/21猪肺泡巨噬细胞的炎症相关细胞因子mRNA转录分析[J]. 西南农业学报, 2016, 29(5):1225-1228.
WANG W, WANG X M, HE K W, et al. Inflammation-associated cytokines mRNA transcriptional profiles of porcine alveolar macrophages cell lines 3D4/21 stimulated by porcine circovirus type 2[J]. Southwest China Journal of Agricultural Sciences, 2016, 29(5):1225-1228. (in Chinese)
[10] AUNG H, SHERMAN J, TARY-LEHMAN M, et al. Analysis of transforming growth factor-beta 1(TGF-β1) expression in human monocytes infected with Mycobacterium avium at a single cell level by ELISPOT assay[J]. J Immunol Methods, 2002, 259(1-2):25-32.
[11] LI Y J, ZOU T, XUE L Y, et al. TGF-β1 enhances phagocytic removal of neuron debris and neuronal survival by olfactory ensheathing cells via integrin/MFG-E8 signaling pathway[J]. Mol Cell Neurosci, 2017, 85:45-56.
[12] PATTI J M, ALLEN B L, MCGAVIN M J, et al. MSCRAMM-mediated adherence of microorganisms to host tissues[J]. Annu Rev Microbiol, 1994, 48:585-617.
[13] BENITO-JARDÍN M, KLAPPROTH S, GIMENO-LLUCH I, et al. The fibronectin synergy site re-enforces cell adhesion and mediates a crosstalk between integrin classes[J]. eLife, 2017, 6:e22264.
[14] WANG B N, LI S Y, SOUTHERN P J, et al. Streptococcal modulation of cellular invasion via TGF-β1 signaling[J]. Proc Natl Acad Sci U S A, 2006, 103(7):2380-2385.
[15] ZHANG B, HE Y B, XU C G, et al. Cytolethal distending toxin (CDT) of the Haemophilus parasuis SC096 strain contributes to serum resistance and adherence to and invasion of PK-15 and PUVEC cells[J]. Vet Microbiol, 2012, 157(1-2):237-242.
[16] LI F Z, LI L B, ZHONG Y, et al. Relationship between LTR methylation and gag expression of HIV-1 in human spermatozoa and sperm-derived embryos[J]. PLoS One, 2013, 8(1):e54801.
[17] LI Y F, ZHANG Y N, XIA Y T, et al. Haemophilus parasuis modulates cellular invasion via TGF-β1 signaling[J]. Vet Microbiol, 2016, 196:18-22.
[18] FRANDOLOSO R, PIVATO M, MARTÍNEZ-MARTÍNEZ S, et al. Differences in Haemophilus parasuis adherence to and invasion of AOC-45 porcine aorta endothelial cells[J]. BMC Vet Res, 2013, 9:207.
[19] ZHANG B, XU C G, ZHANG L Y, et al. Enhanced adherence to and invasion of PUVEC and PK-15 cells due to the overexpression of RfaD, ThyA and Mip in the △ompP2 mutant of Haemophilus parasuis SC096 strain[J]. Vet Microbiol, 2013, 162(2-4):713-723.
[20] KAVANOVÁ L, PRODLALOVÁ J, NEDBALCOVÁ K, et al. Immune response of porcine alveolar macrophages to a concurrent infection with porcine reproductive and respiratory syndrome virus and Haemophilus parasuis in vitro[J]. Vet Microbiol, 2015, 180(1-2):28-35.
[21] FRANDOLOSO R, MARTÍNEZ-MARTÍNEZ S, GUTIÉRREZ-MARTÍN C B, et al. Haemophilus parasuis serovar 5 Nagasaki strain adheres and invades PK-15 cells[J]. Vet Microbiol, 2012, 154(3-4):347-352.
[22] LI N, REN A H, WANG X S, et al. Influenza viral neuraminidase primes bacterial coinfection through TGF-β-mediated expression of host cell receptors[J]. Proc Natl Acad Sci U S A, 2015, 112(1):238-243.

TGF-β1信号通路在副猪嗜血杆菌侵入3D4/21细胞中的作用

TGF-β1 Signaling Stimulates Invasion of Haemophilus parasuis into 3D4/21 Cells

RichHTML

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 12

编辑推荐

Metrics

本文评价

[1]	肖静, 肖坤雪, 王翘楚, 陈焕春, 蔡旭旺, 徐晓娟. 副猪嗜血杆菌Flp-FRT双基因敲除方法的建立与优化[J]. 畜牧兽医学报, 2022, 53(1): 219-230.
[2]	田杨, 刘云宝, 马辉, 潘其聪, 肖静, 陈焕春, 蔡旭旺, 徐晓娟. 副猪嗜血杆菌Apd-ELISA抗体检测试剂盒的制备和初步应用[J]. 畜牧兽医学报, 2020, 51(9): 2227-2237.
[3]	唐梦君, 周倩, 张小燕, 张静, 唐修君, 陆俊贤, 周生, 蒲俊华, 高玉时. 江苏部分地区鸡源与猪源弯曲菌耐药性分析与毒力基因检测[J]. 畜牧兽医学报, 2020, 51(9): 2284-2292.
[4]	程蕾, 陈洪波. 巨噬细胞-γδT细胞免疫轴在猪格拉瑟氏病发病机制中的作用[J]. 畜牧兽医学报, 2018, 49(5): 879-886.
[5]	杨开杰, 冯賽祥, 周琪, 张建民, 廖明, 樊惠英. 副猪嗜血杆菌qseC基因缺失突变对生物被膜形成的影响[J]. 畜牧兽医学报, 2017, 48(8): 1499-1504.
[6]	谨瑾，张禄滑，文心田，李英，代科，周鹏，赵玉佳，曹三杰，黄小波，伍锐，赵勤，文翼平. 副猪嗜血杆菌potD基因缺失株的构建及其部分生物学特性[J]. 畜牧兽医学报, 2016, 47(11): 2274-2279.
[7]	何欢，陈新诺，曾泽，任玉鹏，汤承，张斌，岳华. 副猪嗜血杆菌OmpP2刺激猪肺泡巨噬细胞炎性因子mRNA转录及致炎机制初步分析[J]. 畜牧兽医学报, 2016, 47(7): 1428-1434.
[8]	曾泽，何欢，任玉鹏，岳华，张斌. rfaE基因通过MAPKs/NF-κB信号通路对副猪嗜血杆菌LOS诱导猪肺泡巨噬细胞炎症反应中作用的研究[J]. 畜牧兽医学报, 2016, 47(5): 978-984.
[9]	曾泽，何欢，岳华，汤承，张斌. 副猪嗜血杆菌lgtF基因缺失株的构建及其部分生物学特性[J]. 畜牧兽医学报, 2015, 46(11): 2056-2062.
[10]	曾泽，岳华，冯晓辉，何欢，张斌. rfaE基因在副猪嗜血杆菌LOS刺激猪肺泡巨噬细胞炎性因子mRNA转录中的作用[J]. 畜牧兽医学报, 2015, 46(7): 1232-1237.
[11]	白挨泉，郭建超，覃宗华，蒲文珺，马春全，李国清，张浩吉. 胞内劳森菌TaqMan荧光定量PCR检测方法的建立[J]. 畜牧兽医学报, 2014, 45(10): 1733-1738.
[12]	匡学谦，岳华，陈小飞，汤承. 畜禽空肠弯曲杆菌cmp基因结构特点及ST型研究[J]. 畜牧兽医学报, 2013, 44(4): 657-664.