利用 pdl1 敲除的巨噬细胞转录谱分析结核感染中 PD-L1 作用相关的候选基因
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中国医学科学院医学实验动物研究所;卫健委人类疾病比较医学重点实验室;新发再发传染病动物模型研究 北京市重点实验室;北京市人类重大疾病实验动物模型工程技术研究中心;中国医学科学院结核病中心,北京 100021

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R-33

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Screening of PD-L1 related specific candidate genes in tuberculosis by transcriptome sequencing and analysis in mycobacterium tuberculosis infected pdl1 knockout macrophages
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Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences (CAMS); Key Laboratory of Human Diseases Comparative Medicine, National Health Commission of P.R.C; Beijing Key Laboratory of Animal Models of Emerging and Reemerging Infectious Diseases; Research Center of Laboratory Animal Model Engineering and Technology of Human Critical Diseases in Beijing; Tuberculosis Center, CAMS, Beijing 100021, China

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    摘要:

    目的 分析结核菌(Mtb)感染的程序性死亡配体 1(pdl1)特异性敲除的巨噬细胞(MΦ)转录谱变化,筛选 MΦ 内 PD-L1 作用相关的候选基因。 方法 构建敲除小鼠(pdl1Flox / - ),通过 Cre-loxp 技术培育出 MΦ 上 pdl1 特异性敲除的纯合和杂合小鼠(pdl1Flox / Flox- Cre,pdl1Flox / - - Cre),用 PCR 和流式细胞术验证。 分离上述小鼠 腹腔 MΦ,利用转录组测序技术检测 Mtb 感染 MΦ 24 h 的表达谱,以同窝阴性小鼠(pdl1Flox / Flox)为对照,进行生物信 息学分析。 结果 PCR 和流式细胞术证实小鼠敲除成功。纯合、杂合及同窝阴性小鼠感染前后相比,差异表达基因最显著富集项为参与免疫反应和免疫过程的 GO(P < 0. 01;P < 0. 01),筛出 17 个 PD-L1 相关的候选基因。通过 KEGG 富集分析,最显著富集的通路有 Toll 样受体、NF-κB 信号通路等(P < 0. 01;P < 0. 01),筛出 6 个候选基因。 结论 通过转录谱分析,确定与免疫反应及免疫过程有关的 Ccl2、Qa5、Il12a 等 17 个基因,免疫及炎症相关代谢通 路的 Ptgs2、Cd40、Card11 等 6 个基因为 Mtb 感染的 MΦ 内 PD-L1 相关的候选基因,除 Il6 外均为本研究新筛选的候选基因。

    Abstract:

    Objective To analyze the transcription profile of Mycobacterium tuberculosis ( Mtb )-infected macrophages in which the pdl1 gene has been knocked out and screen PD-L1 related candidate genes in Mtb -infected macrophages (MΦ). Methods pdl1Flox / - mice were constructed, and homozygous (pdl1Flox / Floxwith Cre) and heterozygous (pdl1Flox / - with Cre) mice with targeted inactivation of the pdl1 gene in MΦs were bred by the Cre / loxP technique and verified by PCR and FACS. Peritoneal MΦs were isolated from different substrains of mice, and those isolated from littermate negative mice (pdl1Flox / Flox) were used as controls. Twenty-four hours after infection, the transcript profiles in the infected and uninfected groups were sequenced by RNA-Seq, and bioinformatics analysis was performed. Results pdl1- specific knockout mice were successfully generated and identified by PCR and FACS. The pdl1Flox / Floxwith Cre, pdl1Flox / - with Cre, and pdl1Flox / Flox infected groups were compared with their corresponding non-infected groups. Gene Ontology (GO) functional enrichment analysis showed that the most significant enrichments were immune response (P< 0. 01) and immune system processes (P< 0. 01). The seventeen candidate genes identified were regarded as specific genes related to PD-L1 in Mtb infection. Through the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, the most significantly enriched pathways included Toll-like receptor (P< 0. 01) and nuclear factor kappa-B (NF-κB) (P< 0. 01) signaling pathways, and six candidate genes related to PD-L1 were screened. Conclusions Through transcription analysis, seventeen genes related to the immune response, including Ccl2, Qa5, and Il12a, and six genes in immune - and inflammation-related metabolic pathways, including Ptgs2, Cd40, and Card11, were identified as specific candidate genes related to PD-L1 in macrophages infected with Mtb. Except for Il6, all candidate genes were selected for this research.

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石亚男,唐 军,李军丽,王 杰,占玲俊.利用 pdl1 敲除的巨噬细胞转录谱分析结核感染中 PD-L1 作用相关的候选基因[J].中国比较医学杂志,2020,30(5):31~39.

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  • 收稿日期:2019-12-19
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  • 在线发布日期: 2020-06-19
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