[关键词]
[摘要]
目的:检测萝卜硫素(sulforaphane, SFN)对CD8+T细胞分化、表型及胞内因子分泌的影响并探讨其可能的调控机制。方法:在体外培养实验中,按照SFN处理的剂量分为对照(0 mmol/L)组、SFN 10 mmol/L组、SFN 20 mmol/L组。采用流式细胞术检测SFN对CD8+ T细胞分化、表型及胞内因子分泌的影响以及mTOR siRNA 对CD8+T细胞CD127 和LKRG1 表达的影响;采用qRT-PCR检测抗凋亡因子Bcl-2 和Bcl-6 的表达水平,Annexin-V/PI 双染法检测SFN对CD8+ T细胞凋亡的影响,Western blotting检测信号通路蛋白p-mTOR、p-S6 以及内参b-actin 蛋白的表达。结果:SFN促进CD8+记忆细胞前体细胞的形成、显著降低CD8+T细胞PD-1 和Tim-3 的表达水平(P<0.01)。同时,SFN处理后抗凋亡基因Bcl-2 和Bcl-6 的表达显著增加、CD8+ T细胞的凋亡受到显著抑制,p-mTOR和p-S6 蛋白表达水平也显著降低(P<0.05 或P<0.01)。此外,SFN能够增加促炎性细胞因子IL-2、IFN-g、TNF-a的分泌(P<0.05)。mTOR siRNA 能够显著增加CD127 表达及降低LKRG1 表达水平(均P<0.01)。结论:SFN 可能通过抑制pmTOR信号通路促进CD8+记忆细胞前体细胞的形成,获得更多年轻化的T细胞,为临床免疫细胞治疗提供新的思路。
[Key word]
[Abstract]
Objective: To investigate the effect of sulforaphane (SFN) on CD8+ T cells differentiation, phenotype and the secretion of intracellular cytokines, as well as to study the underlying molecular mechanism. Methods: In the in vitro culture experiment, the cells were categorized into control group, SNF 10 mmol/L group and SNF 20 mmol/L group according to the SNF concentration. The effect of SFN treatment on CD8+ T cells differentiation, phenotype and cytokine secretion were detected by flow cytometry, and the effect of mTOR siRNA on the expression of CD127 and LKRG1 in CD8+T cells was also detected by flow cytometry. Expression of Bcl-2 and Bcl-6 were analyzed by qRT-PCR. The effect of SFN on apoptosis of CD8+T cells was examined by Annexin-V/PI staining. The protein expressions of p-mTOR, p-S6 and b-actin were detected by western blotting. Results: SFN significantly promoted the formation of memory precursor CD8+ T cells and decreased the expression level of PD-1 and Tim-3 in CD8+T cells(P<0.01); meanwhile, after the treatment of SFN, the expressions of anti-apoptosis genes Bcl-2 and Bcl-6 were significantly increased while the apoptosis of CD8+ T cells was significantly inhibited and the protein expressions of p-mTOR and p-S6 were also significantly inhibited(P<0.05 or P<0.01).Moreover, mTOR siRNA could significantly increasethe expression of CD127 and decrease the expression of LKRG1 (all P<0.01).Conclusion: Sulforaphone promotes the formation of memory precursor CD8+T cells possibly by inhibiting the p-mTOR signaling pathway,and this could obtain more T cells to provide new thoughts for clinical immunotherapy.
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[基金项目]
河南省医学科技攻关计划资助项目(No.201702018)