[Study on the gene expression and regulation mechanisms of fibroblasts in acute inflammatory response].

细胞与分子免疫学杂志 Pub Date : 2025-05-01
Meng Du, Hanjing Liao, Manjing Huang, Yaqin Wang, Zongjie Zhao, Zhixiang Zhu, Jun Li
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Abstract

Objective To investigate the gene expression and regulatory mechanisms of mouse embryonic fibroblasts (MEFs) under inflammatory conditions, aiming to elucidate the role of MEFs in inflammatory responses and provide a foundation for discovering anti-inflammatory drugs that act by modulating MEF function. Methods MEFs cultured in vitro were divided into the following groups: lipopolysaccharides (LPS)-treated group, inflammatory conditioned medium (CM)-treated group, and control group, which were treated with LPS, CM, and equal volume solvent, respectively. Transcriptome sequencing was used to analyze the effects of two stimuli on gene expression profile of MEFs. Real time fluorescence quantitative PCR (RT-qPCR) was employed to verify the transcription levels of highly expressed genes of MEFs induced by CM. ELISA was performed to determine the concentrations of cytokines in cell supernatants. Finally, the regulatory effects of CM on the activation of signaling pathways in MEFs were analyzed by immunoblotting. Results Transcriptome analysis showed that both LPS and CM induced the transcription of a large number of genes in MEFs. Compared with LPS, CM potentiated the mRNA transcription of some acute phase proteins, inflammatory cytokines, chemokines, matrix metalloproteinases (MMP), prostaglandin synthetases, and colony-stimulating factors. The transcriptome analysis was verified by RT-qPCR. The results of ELISA showed that CM treatment significantly increased the secretion of interleukin 6 (IL-6), C-C motif chemokine ligand (CCL2), and C-X-C motif chemokine ligand (CXCL1) by MEFs compared with LPS. Mechanism study showed that both LPS and CM induced the phosphorylation of nuclear factor-κB p65 (NF-κB p65), p38 mitogen-activated protein kinase (p38 MAPK), extracellular regulated protein kinases 1/2 (ERK1/2), and TANK-binding kinase (TBK) in MEFs, and CM strongly stimulated the phosphorylation of signal transducer and activator of transcription 3 (STAT3) in MEFs. Conclusion Both LPS and CM can induce transcription and protein secretion of various inflammation-related genes in MEFs. CM can partly enhance LPS-induced activation of MEFs, and the mechanism may be related to the enhancement effect of CM on the activation STAT3 signaling pathway.

[急性炎症反应中成纤维细胞基因表达及调控机制研究]。
目的研究炎症条件下小鼠胚胎成纤维细胞(MEF)的基因表达及其调控机制,旨在阐明MEF在炎症反应中的作用,为发现通过调节MEF功能起作用的抗炎药物提供基础。方法将体外培养的mef分为脂多糖(LPS)处理组、炎症条件培养基(CM)处理组和对照组,分别用LPS、CM和等体积溶剂处理。转录组测序分析了两种刺激对mef基因表达谱的影响。采用实时荧光定量PCR (RT-qPCR)验证CM诱导mef高表达基因的转录水平。ELISA法测定细胞上清液中细胞因子的浓度。最后,通过免疫印迹分析CM对mef中信号通路激活的调控作用。结果转录组分析显示,LPS和CM均诱导了mef中大量基因的转录。与LPS相比,CM增强了一些急性期蛋白、炎症因子、趋化因子、基质金属蛋白酶(MMP)、前列腺素合成酶和集落刺激因子的mRNA转录。转录组分析经RT-qPCR验证。ELISA结果显示,与LPS相比,CM处理显著增加mef分泌白细胞介素6 (IL-6)、C-C基序趋化因子配体(CCL2)和C-X-C基序趋化因子配体(CXCL1)。机制研究表明,LPS和CM均诱导mef中核因子-κB p65 (NF-κB p65)、p38丝裂原活化蛋白激酶(p38 MAPK)、细胞外调节蛋白激酶1/2 (ERK1/2)和罐结合激酶(TBK)的磷酸化,CM强烈刺激mef中信号转导和转录激活因子3 (STAT3)的磷酸化。结论LPS和CM均可诱导mef中多种炎症相关基因的转录和蛋白分泌。CM可以部分增强lps诱导的mef的激活,其机制可能与CM对STAT3信号通路激活的增强作用有关。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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