【作者】 李红；

【导师】 吕全军； 张毅；

【作者基本信息】 郑州大学 ， 营养与食品卫生学（专业学位）， 2018， 硕士

【摘要】 目的恶性肿瘤严重威胁人类健康,是迫切需要解决的公共健康问题。大多数常见恶性肿瘤的预后仍很差,从天然化合物(特别是膳食成分)中寻找安全、有效的抗肿瘤药物是肿瘤治疗领域的研究热点。萝卜硫素是异硫氰酸盐类化合物,被认为是抗肿瘤效果最好的植物活性成分,主要存在于西兰花、卷心菜等十字花科蔬菜。研究表明,萝卜硫素通过不同的分子机制对癌症发生的多个阶段均有抑制作用。流行病学研究也显示常吃西兰花等十字花科蔬菜有助于预防癌症。机体免疫系统在抗肿瘤中发挥重要作用,但萝卜硫素是否通过调控免疫系统发挥抗肿瘤作用尚不清楚。近年来,嵌合抗原受体(chimeric antigen receptor,CAR)T细胞等新型免疫治疗技术治疗肿瘤取得了突破性进展,证实CD8~+T细胞在机体抗肿瘤的关键作用。本课题中,我们拟在体外实验中探索萝卜硫素对CD8~+T细胞表型、功能和分化的影响;并利用识别肿瘤抗原HER2的CAR T(HER2-CAR T)细胞,在体外和小鼠模型中验证萝卜硫素对HER2-CAR T细胞抗肿瘤作用的影响及机制。本研究有助于揭示萝卜硫素抑制肿瘤的新机制,可能为肿瘤免疫治疗提供新手段。方法收集健康志愿者的外周血,分离单个核细胞、用免疫磁珠纯化CD8~+T细胞。用抗CD3/CD28抗体偶联磁珠在体外活化CD8~+T细胞,并分别加入不同浓度的萝卜硫素培养3天;利用流式细胞仪检测CD8~+T细胞表面共刺激分子;利用细胞内因子染色分析CD8~+T细胞分泌的细胞因子;利用Annexin-V/PI染色检测CD8~+T细胞的凋亡,通过定量PCR检测抗凋亡基因;利用流式细胞仪分析CD8~+T细胞分化表型,通过Western blot检测AMPK信号通路磷酸化,并利用siRNA抑制mTOR表达。用流式细胞仪检测人肺癌细胞A549 HER2分子的表达及HER2-CAR T细胞的表型。在体外实验和免疫缺陷小鼠模型中验证萝卜硫素对HER2-CAR T细胞杀伤A549细胞的影响,并分析CD8~+T细胞表型的改变。结果和对照组相比较,萝卜硫素明显下调CD8~+T细胞表面抑制性分子PD-1(20.8±2.92%vs 11.8±2.32%,P=0.0045)和Tim-3的表达(24.0±4.28%vs 14.1±1.38%,P=0.02),但对活化性分子的表达无显著影响;促进CD8~+T细胞分泌IFN-γ(14.20±3.06%vs 30.2±8.88%,P<0.05)、IL-2(10.7±0.99%vs 26.0±6.91%,P<0.05)和TNF-α(14.1±4.18%vs 30.6±4.95%,P<0.03)等细胞因子;显著抑制CD8~+T细胞凋亡(11.3±2.64%vs 5.7±0.78%,P<0.05),上调Bcl-2和Bcl-6等抗凋亡基因的表达;促进CD8~+T细胞分化为前体记忆效应细胞[上调CD127(22.8±4.5%vs 55.5±8.0%,P=0.0064),下调KLRG1(14.2±1.7%vs 3.0±0.6%,P=0.0178)],抑制AMPK信号通路下游分子mTOR及S6的磷酸化。萝卜硫素上调HER2-CAR T细胞表面CD107a,显示对HER2~+A549肺癌细胞更好的杀伤作用;萝卜硫素处理的HER2-CAR T细胞在小鼠体内表现出更好的抑瘤作用,伴有抑制性分子PD-1和Tim-3的表达降低,及记忆相关分子CD127表达增加和KLRG1降低。结论1.萝卜硫素能增强人外周血CD8~+T细胞功能。2.萝卜硫素能促进HER2-CAR T细胞的抗肿瘤作用。3.萝卜硫素为过继免疫细胞治疗治疗肿瘤提供了新思路。更多还原

【Abstract】 ObjectiveMalignant tumors pose a serious threat to human health and are a crucial public health problem.Currently,the prognosis of most common malignant tumors is still poor.Identifying safe and effective anti-tumor medicine from natural compounds,such as dietary substances,is a growing hot topic in the field of cancer therapy.Sulforaphane(SFN)is an isothiocyanate,a phytochemical compound,with outstanding anti-tumor properties,mainly found in broccoli,cabbage and other cruciferous vegetables.Studies have shown that SFN is capable of inhibiting multiple phases of carcinogenesis through various molecular mechanisms.Epidemiological studies have also showed that the regular intake of cruciferous vegetables such as broccoli is beneficial in preventing cancer.The immune system plays a critical role in defending against cancers.However,it is unclear whether SFN inhibits tumor growth through regulating the immune system.Novel cutting-edge techniques such as chimeric antigen receptor(CAR)T cell therapy have made a break-through in clinical application,identifying CD8+T cells as the key effectors in the anti-tumor response.In this study,we investigated the in vitro effects of SFN on CD8+T cells,including phenotypes,functions and differentiation status.Furthermore,we investigated the influence of SFN on the anti-tumor activity of HER2-CAR T in vitro and in vivo using a murine model.In this study we explore a novel anti-tumor mechanism of SFN that may provide a new option for cancer immunotherapy.MethodsBlood samples were collected from healthy donors.Peripheral blood mononuclear cells were isolated and CD8~+T cells were purified by using immune magnetic beads.Anti-CD3/CD28 beads were used to activate CD8~+T cells in vitro,and different concentrations of SFN were added to the culture system for 3 days.The expression of surface costimulatory molecules were analyzed by flow cytometry and the secretion levels of cytokines were detected by intracellular staining.Annexin-V/PI staining was applied to detect the apoptosis of CD8~+T cells,and the expression of anti-apoptosis genes was measured by qRT-PCR.The differentiation status of CD8~+T cells was analyzed by flow cytometry.Western blot was used to detect the phosphorylation of the AMPK signaling pathway,and specific siRNA was used to knockdown mTOR expression.The HER2 expression on human lung cancer A549 cells and the phenotypes of HER2-CAR T cells were analyzed by flow cytometer.The effects of SFN on CD8~+T cell phenotypes and the cytotoxicity of HER2-CAR T cells against A549 cells were tested in vitro and in immunodeficient mice.ResultsSFN dramatically decreased the expression of surface inhibitory molecules such as PD-1(20.8±2.92%vs 11.8±2.32%,P=0.0045)and Tim-3(24.0±4.28%vs 14.1±1.38%,P=0.02),however it had no significant effect on surface activation molecules.Furthermore,SFN increased the expression levels of cytokines such as IFN-γ(14.20±3.06%vs 30.2±8.88%,P<0.05)、IL-2(10.7±0.99%vs 26.0±6.91%,P<0.05)and TNF-α(14.1±4.18%vs 30.6±4.95%,P<0.03).In addition,SFN strongly inhibited activation-induced apoptosis of CD8~+T cells(11.3±2.64%vs5.7±0.78%,P<0.05),with high levels of anti-apoptosis genes Bcl-2 and Bcl-6.SFN promoted CD8~+T cell differentiation into memory precursor effector cells,with upregulated CD127(22.8±4.5%vs 55.5±8.0%,P=0.0064)and downregulated KLRG1(14.2±1.7%vs 3.0±0.6%,P=0.0178).Mechanistically,SFN inhibited the phosphorylation of MAPK signaling downstream molecules,mTOR and S6.SFN could upregulate the expression of CD107a on HER2-CART cells,showing better cytotoxicity against A549 cells in vitro.Finally,SFN-treated HER2-CAR T cells exhibited superior anti-tumor ability in mice,with decreased expression of inhibitory molecules PD-1 and Tim-3,as well as an increase in memory-related molecules and upregulated CD127 and downregulated KLRG1.Conclusion1.SFN can enhance the functions of CD8~+T cells.2.SFN can improve the anti-tumor ability of HER2-CAR T cells.3.SFN may provide a novel strategy to improve adoptive cell therapy.更多还原