摘要
目的探索儿童呼吸道合胞病毒和肺炎衣原体感染所引起的T细胞免疫异常的机制。方法从健康儿童志愿者全血中分离出人外周血单个核细胞,体外实验采用从EDTA抗凝全血中分离的PBMC进行密度梯度离心,并采用抗CD4+的抗体标记磁性微球,通过阳性选择获得特异性细胞型耗竭。从新鲜分离的PBMC中用流式细胞术纯化细胞群。将PBMC悬浮液进行培养,用活病毒或紫外线灭活的呼吸道合胞病毒(RSV)和肺炎衣原体(Cpn)分别进行感染。用特异性抗体对细胞进行表面染色,用FACS CANTOⅡ流式细胞仪对染色样本进行测量,并用FACS DIVA软件进行分析。采用酶联免疫吸附试验(ELISA)对人IFN-γ,IL-10进行定量检测。结果Tregs对RSV感染有反应,在感染后的细胞培养上清液中表现出更高的增殖频率,并上调活化和转运分子的表达。Tregs在RSV感染后的免疫病理限制中起着关键作用。高滴度IgG(≥512)的细胞个体有较强的CMI。RSV感染的细胞中CD4+T细胞IL-10表达增强,并直接抑制CD4+T细胞产生的促炎细胞因子、趋化因子、一氧化氮和前列腺素的表达。IL-10还通过抑制MHCⅡ类和共刺激分子的表面表达,抑制抗原提呈细胞(APC)出现和激活CD4+T细胞的能力。特异性的CD4+T细胞可识别Cpn抗原(细胞质中的或源于MOMP的多肽),在体外可溶解Cpn感染细胞。用Cpn成分做抗原刺激后,对照组较Cpn感染实验组IFN-γ增高。结论在呼吸道合胞病毒(RSV)感染的CD4+T细胞中通过调节细胞因子IL-10表达量引起细胞免疫异常,在肺炎衣原体(Cpn)感染CD4+T细胞中通过调节干扰素IFN-γ引起细胞免疫异常。
Objective To explore the mechanisms of T cell immune abnormality caused by respiratory syncytial virus versus chlamydia pneumoniae infection in children.Methods Human peripheral blood mononuclear cells were isolated from the whole blood of healthy children volunteers.In vitro,PBMC isolated from the whole blood of EDTA anticoagulant was centrifuged with density gradient,and magnetic microspheres labeled with anti-CD4+were used to obtain specific cell type depletion through positive selection.Flow cytometry(FACS)was used to purify cell populations from freshly isolated PBMC.PBMC suspension was cultured and infected with RSV and CPN,respectively.The cell surface was stained with specific antibodies,and the stained samples were measured with FACS cantoⅡflow cytometer,and analyzed with FACS Diva software.Enzyme linked immunosorbent assay(ELISA)was used to detect human IFN-γand IL-10.Results Tregs responded to RSV infection,showed higher proliferation frequency in the supernatant of infected cells,and upregulated the expression of activation and transport molecules.Tregs played a key role in the immunopathological limitation of RSV infection.The cells with a high titer of IgG(≥512)had strong CMI.In RSV infected cells,the expression of IL-10 in the CD4+T cells was enhanced,and the expressions of proinflammatory cytokines,chemokines,nitric oxide and prostaglandins in the CD4+T cells were directly inhibited.IL-10 also inhibited the appearance and activation of the CD4+T cells by inhibiting the surface expression of MHCⅡand costimulatory molecules.The specific CD4+T cells recognized CPN antigens(cytoplasmic or MOMP derived polypeptides)and dissolved the CPN infected cells in vitro.The IFN-γin the control group was higher than that in the experimental group.Conclusions In RSV infected CD4+T cells,IL-10 regulated to cause cellular immune abnormalities.In chlamydia pneumoniae(Cpn)infected CD4+T cells,IFN-γis regulated to cause cellular immune abnormalities.
作者
刘晓霞
陈雪
Liu Xiaoxia;Chen Xue(Yangguang Ronghe Hospital,Weifang 261000,China)
出处
《国际医药卫生导报》
2020年第18期2728-2733,共6页
International Medicine and Health Guidance News
