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q RT-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。
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 q RT-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.
作者
李红
张震
周斌
吕全军
张毅
LI Hong1,2, ZHANG Zhen2, ZHOU Bin2, LYU QuanJun1, ZHANG Yi2(1. College of Public Health; 2. Biotherapy Center of the First Affiliated Hospital, Zhengzhou University, Zhengzhou 450001, Henan, China)
出处
《中国肿瘤生物治疗杂志》
CAS
CSCD
北大核心
2018年第9期920-927,共8页
Chinese Journal of Cancer Biotherapy
基金
河南省医学科技攻关计划资助项目(No.201702018)~~
