氮网球花定碱盐酸盐对活化T细胞增殖的抑制作用

目的探究氮网球花定碱盐酸盐(NMHC)抑制活化T细胞增殖的作用机制。方法Ficoll法提取分离健康人外周血单个核细胞(PBMC),免疫磁珠法纯化出PBMC中的T细胞,抗人CD3/CD28抗体活化纯化后的静息T细胞。NMHC 0.125,0.5,2和8μmol·L^-1与静息T细胞作用,用流式细胞术检测细胞毒性(96 h)、活化T细胞增殖(96 h)、T细胞表面活化标志CD25表达(24 h)和活化T细胞细胞周期(72 h)。NMHC 0.125,0.5,2和8 pmol·L^-1与活化T细胞作用24或48 h,用ELISA测定培养上清中白细胞介素2(IL-2)、IL-6、IL-17A和干扰素γ(IFN-γ)水平。结果NMHC 0.125,0.5,2和8μmol·L^-1抑制抗CD3/CD28抗体活化的T细胞增殖,IC₅₀为(0.28±0.04)μmol·L^-1,且与静息T细胞作用96 h无显著细胞毒性。NMHC显著抑制CD25表达,具有浓度效应关系(r=-0.980,P<0.05)。NMHC 2和8μmol·L^-1作用72 h可调节细胞周期,G₀/G₁期细胞增加(P<0.05),S期细胞减少(P<0.05),具有浓度-效应关系(r=0.920,P<0.05);细胞上清液中IL-2,IL-6,IL-17A和IFN-γ水平显著降低(P<0.05)。结论NMHC可抑制T细胞活化标志分子CD25的表达和IL-2的分泌,阻滞细胞周期于G₀/G₁期,同时抑制炎性细胞因子IL-6,IL-17A和IFN-γ的分泌,这可能是其抑制T细胞增殖的作用机制之一。

Inhibition of N-methylhemeanthidine hydrochloride on activated T cell proliferation

OBJECTIVE To investigate the mechanism by which N-methylhemeanthine hydrochloride(NMHC)inhibits the proliferation of activated T cells.METHODS Healthy peripheral blood mononuclear cells(PBMCs)were extracted and isolated by Ficoll,human peripheral blood T cells were purified by immunomagnetic microbeads from PBMCs,and purified naive T cells were activated by anti-human CD3/CD28 mAbs.Flow cytometry analysis was used to detect the cytotoxicity of naive T cells at 96 h,the proliferation of activated cells at 96 h,the expression of T cell surface activation markers and the cell cycle of activated cells at 72 h with NMHC 0.125,0.5,2 and 8μmol L^-1.The cytokine levels of interleukin-2(IL-2),IL-6,IL-17 A and interferon-γ(IFN-γ)in the cultured supernatant of activated T cells at24 or 48 h were determined by ELISA.RESULTS NMHC obviously inhibited the proliferation of human T cells activated by anti-human-CD3/CD28 mAbs.IC50 was(0.28±0.04)μmol·L^-1,and there was no significant cytotoxicity in naive T cells at 8μmol·L^-1 after 96 h.NMHC significantly inhibited CD25 expression with a concentration-effect relationship(r=-0.980,P<0.05).NMHC 2 and 8μmol·L^-1 arrested cell cycle in G0/G1 phase(P<0.05)for 72 h,and reduced the percentage of S phase cells(P<0.05)also in a concentration-dependent manner(r=0.920,P<0.05).The levels of IL-2,IL-6,IL-17 A and IFN-γsignificantly decreased in the supernatant(P<0.05).CONCLUSION NMHC inhibits the expression of activated T cell marker molecular CD25 and the secretion of IL-2,arrests cell cycle in G0/G1 phase,and inhibits secretion of IL-6,IL-17 A and IFN-y inflammatory cytokines,which may be one of the mechanisms of its inhibition of cell proliferation.