鸟苷酸结合蛋白2b在牛分枝杆菌诱导巨噬细胞极化过程中的作用

doi:10.11843/j.issn.0366-6964.2024.06.035

摘要/Abstract

摘要：

旨在探究鸟苷酸结合蛋白2b (guanylate-binding protein 2b，GBP2b)对M. bovis感染的小鼠单核巨噬细胞系RAW264.7细胞M1、M2型极化的调控及其介导的信号通路。通过小干扰RNA下调RAW264.7巨噬细胞内GBP2b表达，分别利用qRT-PCR、Western blot和流式细胞术检测M1、M2型巨噬细胞表面标志物以及依赖MyD88信号通路关键蛋白表达情况；双萤光素酶报告基因检测GBP2b对NF-κB转录调控。结果表明GBP2b在M. bovis感染的巨噬细胞内高表达.下调RAW264.7巨噬细胞的GBP2b后再经M. bovis感染24 h，M1巨噬细胞表面标志物表达下调，而对M2巨噬细胞表面标志物表达无显著影响。下调GBP2b后抑制了NF-κB的转录启动，依赖MyD88信号通路相关蛋白随GBP2b的下调而下调。结果表明，GBP2b主要通过依赖MyD88信号通路促进M. bovis感染的RAW264.7巨噬细胞向M1表型极化和炎性反应，从而有助于巨噬细胞清除胞内M. bovis。

关键词: 牛分枝杆菌, 鸟苷酸结合蛋白2b, 巨噬细胞, M1极化, M2极化

Abstract:

This study aimed to investigate the effect of guanylate binding protein 2b (GBP2b) on M.bovis infection of RAW264.7 cells and its signal pathways in polarization of M1 and M2 during M.bovis infection. Downregulate the expression of GBP2b in RAW264.7 macrophages through small interfering RNA, and detect the expression of M1 and M2 macrophage surface markers and key proteins related to the MyD88 signaling pathway using qRT-PCR, Western blot, and flow cytometry, respectively. Dual luciferase reporter assay to examine the effect of GBP2b on NF-κB transcriptional regulation. The results showed that GBP2b was highly expressed in macrophages infected with M. bovis. After downregulating the expression of GBP2b in RAW264.7 macrophages and then being infected with M. bovis for 24 h, the expression of surface markers in M1 macrophages was downregulated, while there was no significant effect on the expression of surface markers in M2 macrophages. The transcription initiation of NF-κB is inhibited, and the downregulation of MyD88 signaling pathway related protein expression were due to the downregulation of GBP2b. The results indicate that GBP2b mainly promotes the polarization and inflammatory response of RAW264.7 macrophage infected with M. bovis towards the M1 phenotype by relying on the MyD88 signaling pathway, thereby helping macrophages clear intracellular M. bovis.

Key words: M. bovis, GBP2b, macrophages, M1 polarization, M2 polarization

中图分类号:

S852.4

于有利, 王建东, 郭亚男, 张久盘, 薛峰, 曹钰莹. 鸟苷酸结合蛋白2b在牛分枝杆菌诱导巨噬细胞极化过程中的作用[J]. 畜牧兽医学报, 2024, 55(6): 2641-2651.

Youli YU, Jiandong WANG, Ya'nan GUO, Jiupan ZHANG, Feng XUE, Yuying CAO. Role of Guanylate Binding Protein 2b during Macrophage Polarization Induced by Mycobacterium bovis[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(6): 2641-2651.

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基因名称 Gene names	正义链(5→3′) Sense	反义链(5→3′) Antisense (5′-3′)
siGBP2b-388	GCAGCACCUUCAUCUACAATT	UUGUAGAUGAAGGUGCUGCTT
siGBP2b-765	GACCAGCUGAAUAAAGAAUTT	AUUCUUUAUUCAGCUGGUCTT
siGBP2b-1625	GAGCAACAAAGAAUCAUAUTT	AUAUGAUUCUUUGUUGCUCTT
siCon	UUCUCCGAACGUGUCACGUTT	ACGUGACACGUUCGGAGAATT

基因名称 Gene names	正义链(5′→3′) Sense	反义链(5′→3′) Antisense
GBP2b	GAGTACTCTCTGGAA	TAGATGAAGGTGCTG
iNOS	TGGTGAAGGGACTGAGCTGT	GCTACTCCGTGGAGTGAACA
IL-1β	TGCCACCTTTTGACAGTGATG	TGATGTGCTGCTGCGAGATT
TNF-α	TAGCCCACGTCGTAGCAAAC	GCAGCCTTGTCCCTTGAAGA
Mrc1	ATGGATTGCCCTGAACAGCA	TGTACCGCACCCTCCATCTA
IL-10	CCAAGGTGTCTACAAGGCCA	GCTCTGTCTAGGTCCTGGAGT
Arg1	CTCCAAGCCAAAGTCCTTAGAG	AGGAGCTGTCATTAGGGACATC

抗体 Antibody	来源 Source	稀释比例 Dilution ratio	生产厂家 Manufacture
Anti-B-actin antibody	鼠单克隆抗体	1∶2 000	Cell Signaling
Anti-GAPDH antibody	鼠单克隆抗体	1∶2 000	Cell Signaling
Anti-GBP2b antibody	兔单克隆抗体	1∶500	Cell Signaling
Anti-P-NF-κB p65 antibody	兔单克隆抗体	1∶1 000	Cell Signaling
Anti-NF-κB antibody	兔单克隆抗体	1∶1 000	Cell Signaling
Anti-TLR2 antibody	兔单克隆抗体	1∶1 000	Cell Signaling
Anti-TLR4 antibody	兔单克隆抗体	1∶1 000	Cell Signaling
Anti-MyD88 antibody	兔单克隆抗体	1∶1 000	Cell Signaling
Anti-TRAF-6 antibody	兔单克隆抗体	1∶1 000	Cell Signaling

名称 Name	得分 Score	序列号 Sequence ID	开始 Start	结束 End	有义链 Strand	预测序列 Prediction sequence
MA0105.3NFKB1	7.3007545	GBP2b-WT:	157	167	-	TGGGTTGCCC
MA0105.3NFKB1	5.5649486	GBP2b-WT:	135	145	-	GGGTGTTTCTC
MA0105.1NFKB1	8.029274	GBP2b-WT:	157	166	+	GGGGCAACCC
MA0105.4NFKB1	3.856709	GBP2b-WT:	50	62	-	CGGGGAACCAGCG
MA0105.2NFKB1	8.353432	GBP2b-WT:	29	398	-	CGGGATCCTCC

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