原发免疫性血小板减少症树突状细胞异常免疫反应机制

doi:10.6040/j.issn.1671-7554.0.2022.0069

摘要/Abstract

摘要： 目的探讨影响原发免疫性血小板减少症(ITP)患者树突状细胞功能异常的基因,为ITP治疗寻求新方法。方法随机选取ITP患者(ITP组)8例和同期入院体检健康者8例正常对照组为研究对象,分离外周单个核细胞并在体外诱导分化为单核细胞源性树突状细胞(moDCs),选取ITP组和正常对照组moDCs样本各3例,使用Illumina Hiseq平台进行转录组测序,并完成生物信息学分析。其他moDCs样本分为对照组、ITP组和ITP+雷帕霉素处理组,采用Western blotting法检测哺乳动物雷帕霉素靶蛋白复合体1(mTORC1)信号通路激活情况,采用流式细胞术检测moDCs表面分子表达,采用酶联免疫吸附(ELISA)法检测moDCs细胞因子分泌能力。结果差异性表达分析显示,与正常对照组相比,ITP组患者moDCs中有161个基因表达上调,320个基因表达下调,差异有统计学意义(P<0.05)。利用基因本体论(GO)和京都基因与基因组百科全书(KEGG)数据库对差异基因进行功能和分子通路注释,结果表明差异基因主要集中在T细胞分化、T细胞共刺激、T细胞活化等生物学过程和T细胞受体信号通路等信号途径。基因集富集分析(GSEA)显示,ITP组患者树突状细胞中mTORC1信号通路基因表达上调。进一步验证发现,ITP组患者moDCs中磷酸化哺乳动物雷帕霉素靶蛋白(mTOR)和mTORC1活化标志物磷酸化核糖体蛋白S6激酶(S6K)相对含量升高,同时ITP组患者moDCs共刺激分子CD80、CD86和促炎因子白介素6(IL-6)、白介素12(IL-12)的表达增加,而白介素10(IL-10)、转化生长因子-β(TGF-β)表达降低。使用mTORC1抑制剂雷帕霉素可以抑制moDCs共刺激分子CD80、CD86的表达和促炎因子IL-6、IL-12的分泌,并上调IL-10的表达,而对TGF-β的分泌无明显影响。结论 ITP患者moDCs中mTORC1信号通路高度激活,使用mTORC1抑制剂雷帕霉素可以改善moDCs的免疫调节能力。因此,mTORC1信号通路可能是调节ITP患者moDCs功能异常的新靶点。

关键词: 原发免疫性血小板减少症, 树突状细胞, 转录组测序, mTORC1信号通路, 雷帕霉素

Abstract: Objective To explore the genes affecting the dysfunction of dendritic cells in patients with primary immune thrombocytopenia(ITP), and to seek a new therapeutic approach for ITP. Methods A total of 8 ITP patients and 8 healthy controls were enrolled. Peripheral mononuclear cells were isolated and induced to differentiate into monocyte derived dendritic cells(moDCs)in vitro. The moDCs samples from 3 ITP patients and 3 healthy controls were selected respectively, then transcriptome sequencing was performed using Illumina Hiseq platform, and bioinformatics analysis was completed. The remaining moDCs samples were divided into control group, ITP group and ITP+rapamycin group. The activation of mammalian rapamycin target protein complex 1(mTORC1)signal pathway was detected with Western blotting, the expressions of surface molecules were detected with flow cytometry, and the cytokine secretions were detected with enzyme-linked immunosorbent assay(ELISA). Results Differential gene analysis showed that compared with the healthy controls, ITP patients had 161 significantly up-regulated genes and 320 significantly down-regulated genes(P<0.05). The differential genes were mainly concentrated in biological processes such as T cell differentiation, T cell costimulation, T cell activation and T cell receptor signaling pathway. Gene set enrichment analysis(GSEA)showed that the activity of mTORC1 signal pathway in moDCs of ITP patients was enhanced. Further validation found that the expression of phosphorylated mTOR and mTORC1 activation marker phosphorylated ribosomal protein S6 kinase(S6K)in moDCs of ITP patients were increased. The expressions of moDCs costimulatory molecules CD80 and CD86, and pro-inflammatory factors interleukin(IL)-6 and IL-12 were increased in ITP patients, while the expressions of IL-10 and transforming growth factor-β(TGF-β)were decreased. The mTORC1 inhibitor rapamycin inhibited the expressions of CD80, CD86, IL-6 and IL-12. Rapamycin upregulated the expression of IL-10, but did not affect the expression of TGF-β. Conclusion The mTORC1 signaling pathway is highly activated in moDCs of ITP patients, and the use of rapamycin can improve the immunomodulatory ability of moDCs. Therefore, mTORC1 signaling pathway may be a new target for regulating moDCs dysfunction in ITP patients.

Key words: Primary immune thrombocytopenia, Dendritic cells, Transcriptome sequencing, mTORC1 signaling pathway, Rapamycin

中图分类号:

R558

相宇娇,刘强,刘璐,石艳. 原发免疫性血小板减少症树突状细胞异常免疫反应机制[J]. 山东大学学报 (医学版), 2022, 60(7): 89-97.

XIANG Yujiao, LIU Qiang, LIU Lu, SHI Yan. Mechanism of abnormal immune response of dendritic cells in immune thrombocytopenia[J]. Journal of Shandong University (Health Sciences), 2022, 60(7): 89-97.

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