| 摘要: |
| 【 目的】揭示不同免疫状态下感染布鲁氏菌（布菌）对小鼠CD4+T细胞基因表达的影响。【 方法】将小鼠随机分为对照组（CK）、布菌M5+gfp组（M5+gfp组）、环磷酰胺免疫抑制组（CYP 组）、CYP+布菌M5+gfp组（CYP+M5+gfp组），测定小鼠脾组织相对质量、载菌量，HE染色评估脾脏病理变化；测定半数溶血值以及 IFN-γ 含量，综合评价模型小鼠机体免疫状态。对4种模型小鼠的CD4+T细胞进行转录组测序，对其差异表达基因进行KEGG分析。选取差异基因Klf4、Elane、Fosb、Fcgr1、Chtf18、Gadd45g进行RT-qPCR验证。【 结果】① M5+gfp组小鼠脾组织相对质量显著高于CK组（P<0.05），CYP+M5+gfp组极显著高于CYP组（P<0.01）。CYP+M5+gfp组载菌量极显著高于M5+gfp组（P<0.01）。M5+gfp组和CYP+M5+gfp组脾组织细菌PCR均扩增出1 000 bp的GFP片段，证实侵染脾组织的菌种为布菌M5+gfp菌株。HE染色结果显示，M5+gfp组和CYP+M5+gfp组脾脏均呈现红白髓分界不清、脾结节减少及淋巴细胞稀疏等病理变化，其中CYP+M5+gfp组病变程度更重。半数溶血值测定结果显示，M5+gfp组低于CK组，CYP+M5+gfp组低于CYP组，但差异均不显著（P>0.05）。IFN-γ含量测定结果显示，M5+gfp组显著高于CK组（P<0.05），CYP+M5+gfp组极显著高于CYP组（P<0.01）。② M5+gfp组较CK组CD4+T细胞上调基因富集于抗原加工和呈递、NOD样受体信号通路，可增强机体对布菌的清除能力；下调基因富集于自然杀伤细胞介导的细胞毒性通路，可导致NK细胞功能受损，削弱机体抗布菌能力。CYP+M5+gfp组较CYP组CD4+T细胞上调基因富集于细胞因子细胞因子受体相互作用、中性粒细胞胞外陷阱形成及造血细胞谱系通路，可协同强化机体免疫能力；下调基因富集于细胞因子-细胞因子受体相互作用通路，可导致免疫信号受到干扰。筛选出M5+gfp组较CK组和CYP+M5+gfp组较CYP组均上调的共有基因56个，其中Clca3a1、Trbv19、Gbp2b、Klf1、Serpina3f、Trbv17在M5+gfp组和CYP+M5+gfp组小鼠中表达量较高。Klf4、Elane、Fosb、Fcgr1、Chtf18、Gadd45g基因表达水平转录组测序结果与RT-qPCR验证结果 吻合，证实转录组测序数据是可靠的。【 结论】布菌感染期间，小鼠CD4+T细胞基因表达模式发生了改变；筛选出56个不同免疫状态下均上调的核心基因。 |
| 关键词: 布鲁氏菌 CD4+T细胞 免疫抑制 转录组测序 差异表达基因 |
| DOI：10. 13207/j. jnwafu. 2026. 09. 009 |
| 分类号: |
| 基金项目:国家重点基础研究发展计划项目（2010CB530200） |
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| Effects of Brucella melitensis on gene expression in CD4+T cells of mice with different immune statuses |
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JIN Jing, WANG Kang, GENG Fanghao, LI Yi, GAO Jianfeng
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College of Life Sciences，Shihezi University，Shihezi， Xinjiang 832000，China
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| Abstract: |
| 【 Objective】This research aims to elucidate the effects of Brucella melitensis infection on CD4+T cell gene expression in mice under different immune statuses.【Method】Mice were randomly divided into the control group（CK group），Brucella melitensis M5+gfp group（M5+gfp group），cyclophosphamide immunosuppres?sion group（CYP group）， and CYP+Brucella melitensis M5+gfp group（CYP+M5+gfp
group）.Relative mass and bacterial load of mouse spleen tissue were determined，and HE staining was used to assess the pathological changes in the spleen tissue. The half-hemolytic complement activity（CH50） and IFN-γ levels were determined to comprehensively evaluate the immune status of the model mice. Transcriptome sequencing analysis was performed on CD4+T cells from the four groups，and KEGG analysis was performed on differentially expressed genes. The differentially expressed genes Klf4、Elane、Fosb、Fcgr1、Chtf18 and Gadd45g were selected for RT-qPCR validation.【Result】① The relative mass of spleen tissue in the M5+gfp group was significantly higher than that in the CK group（P<0.05），and that in the CYP+M5+gfp group was significantly higher than that in the CYP group（P<0.01）.The bacterial load in the CYP+M5+gfp group was extremely significantly higher than that in the M5+gfp group（P<0.01）.1 000 bp GFP fragments amplified in bacterial PCR of spleen tissues were obtained both in the M5+gfp and CYP+M5+gfpgroups，confirming that the bacterial strain infecting the spleen tissue was the M5+gfp strain of Brucella melitensis. HE staining results showed that pathological changes such as unclear red-white marrow demarcation，reduced spleen nodules，and sparse lymphocytes were observed in both the M5+gfp group and the CYP+M5+gfp group，with the lesions being more severe in the CYP+ M5+gfpgroup. The half-hemolytic complement activity measurement results showed that the half value of hemolysin in the M5+gfp group was lower than that in the CK group，and that in the CYP+M5+gfp group was lower than that in the CYP group，but the differences were not significant（P>0.05）.IFN-γ level in the M5+gfp group were significantly higher than that in the CK group（P<0.05），and that in the CYP+M5+gfp group was extremely significantly higher than that in the CYP group（P<0.01）.② Compared with the CK group，the up-regulated genes of CD4+T cells in the M5+gfp group were enriched in antigen processing and presentation and NOD-like receptor signaling pathways，enhancing the host’s clearance of Brucella melitensis. The down-regulated genes were enriched in the natural killer cell-mediated cytotoxic pathway， which impaired NK cell function and weakened the antibacterial ability against Brucella melitensis. Compared with the CYP group，the up-regulated genes of CD4+T cells in the CYP+M5+gfp group were enriched in cytokine-cytokine receptor interaction，neutrophil extracellular traps，and hematopoietic cell lineage，synergistically enhancing the host’s immune capa-city.The down-regulated genes were enriched in the cytokine-cytokine receptor interaction pathway，which led to interference with immune signaling. A total of 56 genes were identified that were upregulated in both the M5+gfp group compared to the CK group and the CYP+M5+gfpgroup compared to the CYP group. Among them，Clca3a1、Trbv19、Gbp2b、Klf1、Serpina3f and Trbv17 exhibited higher expression levels in the M5+gfp and CYP+M5+gfp model mice. In addition，the transcriptomic sequencing data of Klf4、Elane、Fosb、Fcgr1、Chtf18 and Gadd45g genes were consistent with the RT-qPCR validation results，confirming the reliability of the tran-scriptomic sequencing data.【Conclusion】During Brucella melitensis infection，the gene expression patterns of CD4+T cells in mice changed，and ultimately 56 core genes that were upregulated under different immune conditions were selected. |
| Key words: Brucella melitensis CD4+T cells immunosuppression transcriptome sequencing differentially expressed gene |
