类风湿关节炎患者 iNKT 细胞频率与 IFN-γ／IL-4的相关性研究

Study of the correlation between the percentage of iNKT cells and the ratio of IFN-γ/IL-4 in patients with rheumatoid arthritis

目的：检测类风湿关节炎患者外周血中恒定自然杀伤T细胞（ invariant nature killer T， iNKT）的频率及功能的变化，分析iNKT细胞频率与血清中IFN-γ／IL-4比值的相关性，进一步明确iNKT细胞在类风湿关节炎（ rheumatoid arthritis， RA）发病中的意义。方法收集70例RA患者（并对其中30例患者在缓解期连续跟踪）和40例健康人外周静脉抗凝血并分离单个核细胞（ peripheral blood mononuclear，PBMC），采用流式细胞技术（fluorescence-activated cell sorting，FACS）分析样本中iNKT细胞频率；再对PBMC体外培养，FACS动态检测iNKT细胞增殖频率；MILLIPLEX MAP Human Cytokine／Chemokine kit检测相应血清及PBMC培养上清中IFN-γ／IL-4的水平；RT-PCR检测iNKT细胞中IFN-γmRNA与IL-4 mRNA表达水平。结果与健康对照组相比，RA患者iNKT细胞频率及增殖频率显著降低（P＜0．05），增殖延迟。治疗后缓解期患者iNKT细胞频率及增殖频率有所恢复，与活动期RA患者比较差异有统计学意义（P＜0．05），与健康对照组比较差异无统计学意义（P＞0．05）；与健康对照组相比，活动期RA患者血清及PBMC培养上清中IFN-γ／IL-4比值升高，差异有统计学意义（ P＜0．05）。缓解期RA患者血清中IFN-γ／IL-4比值下降，与活动期比较差异有统计学意义（ P＜0．05），与健康对照组比较，差异无统计学意义（P＞0．05）；活动期RA患者iNKT细胞中IFN-γmRNA表达升高、IL-4 mRNA表达降低。缓解期RA患者与健康对照组间IFN-γmRNA、IL-4 mRNA表达水平的差异不明显；RA患者外周血中iNKT细胞频率与IFN-γ／IL-4比值呈负相关（ P＜0．05）。结论RA患者存在iNKT细胞频率低下与功能缺陷。 iNKT细胞参与RA发病过程，且与病情活动性密切相关。

Objective To investigate the alterations of invariant nature killer T（ iNKT） cells in peripheral blood samples from patients with rheumatoid arthritis （ RA） and to clarify the correlation between the percentage of iNKT cells and the ratio of IFN-γ/IL-4 in order to further understand the significance of iNKT cells in the development of RA.Methods Peripheral blood mononuclear cells （ PBMCs） were isola-ted from 70 patients with RA and 40 healthy subjects.Among them, thirty patients in the stage of inactive RA were involved in a follow-up study.Fluorescence activated cell sorting （ FACS） was used to detect the percentage of iNKT cells.PBMCs were cultured in vitro for analysis of cytokine production.The dynamic changes of iNKT cells in percentages were analyzed by FACS.MILLIPLEX MAP Human Cytokine/Chemo-kine kit was used to measure the secretion of IFN-γand IL-4 in serum samples and culture media of PBMCs. The expression of IFN-γand IL-4 in iNKT cells at mRNA level were analyzed by RT-PCR.Results Com-pared with the healthy subjects, the patients with active RA showed the delayed proliferation of iNKT cells and the decreased percentages and proliferation rates of iNKT cells （P〈0.05）.The percentages and prolif-eration rates of iNKT cells in patients with active RA were significantly lower than those in patients with inac-tive RA （P〈0.05）.No statistical significant differences with iNKT cells were found between healthy sub-jects and patients with inactive RA （P〉0.05）.The ratios of IFN-γ/IL-4 in serum samples and culture media of PBMCs were increased in patients with active RA as compared with those in patients with inactive＆amp;nbsp;RA and healthy subjects （P〈0.05）.No statistical significant differences with the ratios of IFN-γ/IL-4 were observed between healthy subjects and patients with inactive RA （P〉0.05）.Compared with healthy subjects and patients with inactive RA, patients with active RA showed increased transcriptional level of IFN-γand decreased transcriptional level of IL-4.No significant differences with the expression of IFN-γand IL-4 in iNKT cells at mRNA level were observed between healthy subjects and patients with inactive RA.The per-centage of iNKT cells was negatively related to the IFN-γ/IL-4 ratio in patients with RA （P〈0.05）.Con-clusion Decreased percentage and impaired function of iNKT cells were detected in patients with RA. iNKT cells were closely related to the development and disease activity of RA.