摘要
目的:探讨萝卜硫素(SFN)对靶向人类表皮生长因子受体-2(HER2)的嵌合抗原受体修饰T细胞(CART)分化、细胞内因子分泌、抗肿瘤能力的影响。方法:采用密度梯度离心法及细胞免疫磁珠分选健康供者外周血CD8^+ T细胞,制备HER2 CAR-T细胞,用15μmol/L SFN处理72 h后检测其分化情况。将HER2 CAR-T细胞与A549共孵育,检测HER2 CAR-T细胞内IFN-γ和TNF-α的表达及A549细胞的凋亡,采用qRT-PCR检测细胞糖酵解途径相关酶[己糖激酶2(HK2)、磷酸果糖激酶(PFKM,PFKP)、丙酮酸激酶(PKM)]及脂肪酸氧化过程相关酶[肉毒碱棕榈酰基转移酶(CPT1A)]mRNA的表达。以未经SFN处理的细胞作为对照。结果:与对照组相比,SFN组中心记忆性T细胞亚群比例增加(P <0. 05),IFN-γ及TNF-α分泌水平增加,对A549细胞的杀伤能力增强(P <0. 05),HK2、PFKM、PFKP和PKM mRNA表达下调,CPT1A mRNA表达上调(P <0. 05)。结论:SFN通过抑制糖酵解途径促进HER2 CAR-T细胞向记忆亚群分化,同时增强其抗肿瘤效应。
Aim:To explore the effects of sulforaphane(SFN)on differentiation,cytokine secretion and anti-tumor function of HER2 CAR-T cells.Methods:CD8^+T cells were isolated by density gradient centrifugation and magnetic beads from peripheral blood of healthy donors.Then we constructed HER2 CAR retrovirus vector to transfect CD8^+T cells and analysed the differentiation of HER2 CAR-T cells treated with 15μmol/L SFN after 72 hours.A549 cells were cocultured with HER2 CAR-T cells and flow cytometer was used to detect the intracellular cytokines(IFN-γand TNF-α)secretion level of HER2 CAR-T cells and apoptosis of tumor cells.The expression of glycolysis and fatty acid oxidation(FAO)related genes were detected by qRT-PCR.The cells not treated by SFN were the control.Results:Compared with control group,the proportion of central memory T cells of SFN-treated HER2 CAR-T cells was up-regulated(P<0.05).After cocultured with tumor cells,the levels of IFN-γand TNF-αsecreted by SFN-treated HER2 CAR-T cells were increased(P<0.05),and apoptosis of tumor cells was also increased(P<0.05).In SFN-treated group,the genes related to glycolysis(HK2,PFKM,PFKP,PKM)were down-regulated while the gene related to FAO(CPT1A)was up-regulated(P<0.05).Conclusion:SFN promotes the differentiation of HER2 CAR-T cells into memory subpopulations and anti-tumor ability via inhibiting the glycolytic pathway.
作者
申春一
张震
田永贵
张毅
SHEN Chunyi;ZHANG Zhen;TIAN Yonggui;ZHANG Yi(Biotherapy Center,the First Affiliated Hospital,Zhengzhou University,Zhengzhou 450052;Cancer Center,the First Affiliated Hospital,Zhengzhou University,Zhengzhou 450052;Henan Key Laboratory for Tumor Immunology and Biotherapy,Zhengzhou 450052)
出处
《郑州大学学报(医学版)》
CAS
北大核心
2019年第3期340-344,共5页
Journal of Zhengzhou University(Medical Sciences)
基金
国家自然科学基金资助项目(81771781
U1804281
81802857)
国家重点研发计划重点专项资助项目(2018YFC1313400)
河南省重大科技专项资助项目(161100311000)