Preliminary research on the expression levels of PD-1 on memory T cells in the peripheral blood from T1DM patients with the development and progression of disease
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摘要:
目的：通过检测1型糖尿病（type 1 diabetes mellitus，T1DM）、2型糖尿病（type 2 diabetes mellitus，T2DM）患者以及健康对照外周血T细胞亚群免疫负调控分子程序性死亡蛋白1（programmed cell death protein 1，PD-1）mRNA相对表达水平和记忆性T细胞（memory T cells，Tm）表面PD-1表达情况，从分子生物学、细胞免疫学等多种角度研究记忆性T细胞PD-1表达异常与以胰岛β细胞破坏为主要机制的T1DM发生发展的相关性。方法：分离T1DM、T2DM患者以及健康对照组外周血单个核细胞（peripheral blood mononuclear cells，PBMCs）；从PBMCs中分离出CD4+T细胞和CD8+T细胞，使用荧光定量PCR分别检测其细胞内PD-1 mRNA相对表达水平；将PBMCs分别标记荧光抗体CD4-FITC、CD45RO-PE、CCR7-APC、CD8-FITC、PD-1-PerCp，应用流式细胞术分别检测CD4+CD45RO+CCR7+Tcm细胞、CD4+CD45RO+CCR7-Tem细胞、CD8+CD45RO+CCR7+Tcm细胞、CD8+CD45RO+CCR7-Tem细胞表面免疫负调控分子PD-1的表达水平。结果：①T1DM组患者外周血中CD4+T细胞内PD-1 mRNA相对表达水平低于T2DM组和正常对照组，差异有统计学意义；②T1DM组患者外周血中CD8+T细胞内PD-1 mRNA相对表达水平未见明显异常；③T1DM组患者外周血中CD4+CD45RO+CCR7-Tem细胞亚群与CD4+CD45RO+CCR7+Tcm细胞亚群PD-1表达水平均显著低于正常对照组和T2DM组患者，差异有统计学意义；④T1DM组患者外周血中CD8+CD45RO+CCR7-Tem细胞亚群与CD8+CD45RO+CCR7+Tcm细胞亚群PD-1表达水平均无明显异常。结论：胰岛β细胞可通过细胞表面PD-L1与CD4+Tm细胞表面PD-1结合从而负调节后者细胞免疫作用，因此当CD4+Tm细胞PD-1表达异常而失去负调控作用时，细胞效应则会进一步增强，最终可能通过破坏胰岛β细胞而导致T1DM的发生发展。
Abstract:
Objective：Through the detection of the relative abundance of immune negative control molecules programmed cell death protein 1（PD-1） mRNA in T-cell subsets and the PD-1 expression on memory T cells from peripheral blood in type 1 diabetes mellitus（T1DM） patients，type 2 diabetes mellitus（T2DM） patients and healthy control separately，to further study the relationship of abnormal expression of PD-1 on memory T cells with the development and progression of T1DM which is characterized by pancreatic β-cell destruction. Methods：Peripheral blood mononuclear cells（PBMCs）were isolated from health control，T1DM patients，and T2DM patients separately；PBMCs were further sorted into CD4+T cells and CD8+T cells，then the relative abundance of PD-1 mRNA was analyzed by fluorescence quantitative PCR；PBMCs were marked by fluorescent antibody CD4-FITC，CD45RO-PE，CCR7-APC，CD8-FITC and PD-1-PerCp separately，then analyzed the expression of PD-1 on CD4+CD45RO+CCR7+Tcm cells，CD4+CD45RO+CCR7-Tem cells，CD8+CD45RO+CCR7+Tcm cells and CD8+CD45RO+CCR7-Tem cells by flow cytometry（FCM）. Results：①The relative abundance of PD-1 mRNA in CD4+T cells from the peripheral blood of the T1DM group was significantly lower than that of the T2DM group and the healthy control group；②There was no abnormality for the relative abundance of PD-1 mRNA in CD8+T cells from the peripheral blood of the T1DM group，compared with the T2DM and healthy control groups；③The expression levels of PD-1 on CD4+CD45RO+CCR7-Tem cells and CD4+CD45RO+CCR7+Tcm cells in the peripheral blood from T1DM patients were significantly lower than those of the healthy control and T2DM group；④There was no significant difference for the expression of PD-1 on CD8+CD45RO+CCR7-Tem cells and CD8+CD45RO+CCR7+Tcm cells between the T1DM group，the T2DM group and the healthy control group. Conclusion：For PD-L1 which is expressed on islet β cells can combine with PD-1 on CD4+Tm cells to negatively regulate cell immunity，so when the expression of PD-1 on CD4+Tm cells is abnormal，cell effect will be further enhanced for the loss of negative regulation，so it may eventually lead to the development of T1DM by destroying islet β cells.
