新型杂二萜类化合物Sis-25抑制T细胞增殖机制研究

发布时间：2018-06-19 11:30

本文选题：Sis-25 + T细胞　；参考：《成都医学院》2017年硕士论文

【摘要】：免疫抑制剂在临床上被广泛应用于治疗自身免疫性疾病及器官移植排斥反应。然而这些疾病属于多因素诱发、多组织器官参与的复杂性疾病,患者的个体差异及药物敏感性的不同,导致单独使用一种免疫抑制剂,如环孢霉素、他克莫司、雷帕霉素、霉酚酸酯、FTY720等,不能完全控制病情,还需采用联合、轮换或序贯治疗。因此,继续寻找和开发新型免疫抑制剂将为患者提供更多的选择。来源于天然产物的药物治疗疾病的历史悠久,天然产物资源丰富、毒副作用小,因此将其开发成为免疫抑制剂具有十分显著的优势。从帚状香茶菜分离获得具有新颖骨架杂二萜类化合物Scopariusicides A,发现Scopariusicides A可显著抑制抗CD3/CD28抗体诱导的人T细胞增殖,提示其具有免疫抑制活性,通过对它生源关系的探讨,设计并完成Scopariusicides A的仿生合成,结构修饰后得到一系列结构类似的衍生物,经免疫抑制活性筛选,我们发现有化合物Sis-25具有最强的免疫抑制活性。本论文从体外细胞分子水平上探索Sis-25抑制T细胞增殖的作用机制,为后续开发成为应用于临床的新型免疫抑制剂提供理论基础与实验依据。目的从人外周血单个核细胞(peripheral blood mononuclear cell,PBMC)分离纯化获得T细胞,并对其进行鉴定及增殖特性研究。在此基础上探索新型杂二萜类化合物Sis-25抑制T细胞增殖的作用机制。方法(1)采用Ficoll密度梯度离心法分离人外周血获取外周血单个核细胞,再经免疫磁珠纯化得到T细胞。(2)流式细胞术检测细胞增殖、细胞凋亡、细胞活力、CD25和CD69表达以及细胞周期。(3)酶联免疫吸附法(enzyme linked immunosorbent assay,ELISA)检测促性细胞因子IL-6、IL-17和IFN-γ,以及抗炎细胞因子IL-10的分泌。(4)蛋白免疫印迹法(Western blotting)检测GSK3β、p70S6K、Akt、STAT5和ERK1/2的蛋白表达和磷酸化。结果本研究成功分离纯化获得T细胞,并使T细胞纯度达到95%以上。根据T细胞生长相对曲线可知,在72hr至96hr这一时间段是T细胞对数生长期,这一时间段可作为化合物免疫抑制活性检测观察最佳时间点。在此基础上我们发现,Sis-25可显著抑制抗CD3/CD28抗体诱导的人T细胞增殖及混合T淋巴细胞反应,IC50分别为5.5±0.7μM和5.1±1.1μM。Sis-25对抗人CD3/CD28抗体诱导活化人T细胞发生细胞凋亡没有影响,并且对初始静息T细胞、IL-4处理的活化T细胞(此细胞可维持活化状态但无增殖能力)以及静息的PBMC无显著细胞毒性,提示Sis-25选择性地抑制T细胞活化增殖。Sis-25不抑制T细胞活化表面标志物CD25和CD69表达以及细胞因子IL-2分泌,提示Sis-25不作用T细胞活化阶段,其抑制T细胞增殖的作用机制不同于FK506。Sis-25阻滞活化的T细胞周期于G0/G1期,且呈剂量依赖效应。Sis-25显著抑制活化T细胞分泌炎性细胞因子IL-6,IL-17A和IFN-γ,但不影响抗炎因子IL-10的分泌。Sis-25显著抑制IL-2诱导活化T细胞增殖(IC50:4.5±0.9μM)与p70S6K磷酸化,但不影响Akt、STAT5与ERK1/2蛋白表达及其磷酸化,并且可增强GSK3β蛋白磷酸化。结论综上所述,Sis-25可能是通过阻断mTOR/p70S6K信号通路达到抑制人T细胞增殖。Sis-25有望作为免疫抑制剂先导化合物,为开发治疗自身免疫性疾病及器官排斥反应的新型免疫抑制剂提供前期的理论基础与实验依据。
[Abstract]:Immunosuppressants are widely used in the clinical treatment of autoimmune diseases and organ transplant rejection. However, these diseases are caused by multiple factors, complex diseases involving multiple organs, individual differences in patients and different drug sensitivity, resulting in the use of an immunosuppressant, such as cyclosporin, and tacrolimus alone. Rapamycin, mycophenolate mofetil, FTY720, and so on, can not completely control the disease, but also need to be combined, rotated or sequential therapy. Therefore, continuing to find and developing new immunosuppressive agents will provide more options for patients. Scopariusicides A, a novel two terpenoid compound with novel skeleton, was obtained from broom like tea. It was found that Scopariusicides A could significantly inhibit the proliferation of T cells induced by anti CD3/CD28 antibody, suggesting that it has immunosuppressive activity. The bionic synthesis of Scopariusicides A was completed. After the structural modification, a series of analogous derivatives were obtained. After screening the immunosuppressive activity, we found that compound Sis-25 had the strongest immunosuppressive activity. This paper explored the mechanism of Sis-25 inhibition of T cell proliferation from the cell molecular level in vitro, and was used for subsequent development. A new clinical immunosuppressant provides theoretical basis and experimental basis. Objective to isolate and purify T cells from human peripheral blood mononuclear cells (peripheral blood mononuclear cell (PBMC)), and to identify and proliferate properties of T cells. Based on this, a new type of hetero two terpenoids, Sis-25, is explored to inhibit the proliferation of T cells. Methods (1) Ficoll density gradient centrifugation was used to separate peripheral blood mononuclear cells from human peripheral blood and T cells were purified by immunomagnetic beads. (2) flow cytometry was used to detect cell proliferation, cell apoptosis, cell viability, CD25 and CD69 expression and cell cycle. (3) enzyme linked immunosorbent assay (enzyme linked immunosorbent assay, ELISA) IL-6, IL-17 and IFN- gamma, and the secretion of anti-inflammatory cytokine IL-10 were detected. (4) protein expression and phosphorylation of GSK3 beta, p70S6K, Akt, STAT5 and ERK1/2 were detected by protein immunoblotting (Western blotting). Results the T cells were isolated and purified, and the purity of the cells was above 95%. The curve shows that the time period of 72hr to 96hr is the logarithmic growth period of T cells. This time period can be used as the best time to detect the immunosuppressive activity of compounds. On this basis, we found that Sis-25 can significantly inhibit the proliferation of CD3/CD28 antibody induced human T cells and the reaction of mixed T lymphocyte, IC50 is 5.5 + 0.7 u M and 5.1 + respectively. 1.1 mu M.Sis-25 did not affect the apoptosis of activated human T cells induced by human CD3/CD28 antibody, and there was no significant cytotoxicity for the initial resting T cells, IL-4 treated activated T cells (the cells could maintain the activation state but no proliferation ability) and the resting PBMC had no significant cytotoxicity, suggesting that Sis-25 selectively inhibited the T cell activation and proliferation of.Sis-25. The expression of T cell activation surface markers, CD25 and CD69, and the secretion of cytokine IL-2, suggesting that Sis-25 does not act on the activation phase of T cells. The mechanism of its inhibition of T cell proliferation is different from that of the T cell cycle activated by FK506.Sis-25 block in G0/G1 period, and the dose dependence effect.Sis-25 significantly inhibits the activation of inflammatory cytokines secreted by the activated T cells. 6, IL-17A and IFN- gamma, but does not affect the secretion of.Sis-25 by the anti-inflammatory factor IL-10 significantly inhibits the proliferation of IL-2 induced activated T cells (IC50:4.5 + 0.9 mu M) and p70S6K phosphorylation, but does not affect the expression and phosphorylation of Akt, STAT5 and ERK1/2 protein, and can enhance the phosphorylation of the beta protein. The pathway to inhibit the proliferation of human T cells is expected to be a precursor of immunosuppressive agents. It provides the theoretical basis and experimental basis for the development of new immunosuppressive agents for the treatment of autoimmune diseases and organ rejection.
【学位授予单位】：成都医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R96

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