微生物来源的天然产物的免疫抑制活性的筛选及评价

	微生物来源的天然产物的免疫抑制活性的筛选及评价; Screening and Evaluation of Microbial-derived Natural Products with Immunosuppressive Activity
	周知
导师	黄胜雄
摘要	Immunosuppressive agents, a class of drugs inhibits the immune response, are widely used in organ transplantation to reduce rejection. Immunosuppressive agents have been used for a variety of autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, and psoriasis. In this study, the immunosuppressive activity of microbial secondary metabolites was screened by in vitro screening model, moreover, the mechanism of action was explored preliminarily. Objective T cells were isolated and purified from healthy human peripheral blood to study their proliferation characteristics in vitro. Screening natural products with immunosuppressive activity from microbial secondary metabolites by in vitro screening model, preliminary study on its mechanism of inhibiting T cell proliferation, and providing lead compounds for the development of novel immunosuppressants Method Peripheral blood mononuclear cells (PBMC) were isolated from healthy human peripheral blood by Ficoll density gradient centrifugation, and then subjected to magnetic bead cell sorting (MACS) to obtain human T cells. Flow cytometry combined with fluorescence staining was used to detect cell proliferation, cell viability, apoptosis, cell cycle and CD25 expression. Enzyme-linked immunosorbent assay (ELISA) was used to detect the secretion of cytokines IL-2, IL-6 and IL-17A. Western blotting (WB) was used to detect the expression and phosphorylation of STAT5, STAT3, p70S6K, Akt and ERK1/2. Result Human T cells that the purity was greater than 97% can be isolated and purified from healthy human peripheral blood by Ficoll density gradient centrifugation and MACS. The in vitro growth curve of T cells showed a peak of proliferation at 96 h after stimulation. Therefore, the time to detect the immunosuppressive activity of the compound was 96 hours after the stimulation. Through this in vitro model, four natural products with immunosuppressive activity were screened from secondary metabolites of microorganisms, of which Sanqimycin E had the best activity. The results showed that Sanqimycin E significantly inhibited T cell proliferation induced by anti-human CD3/CD28 antibody stimulation and PHA stimulation with IC50 values of 9.3±1.2 μM and 9.2±2.5 μM, respectively, which did not cause significant apoptosis in T cells stimulated by anti-human CD3/CD28 antibody, and has no significant cytotoxicity to resting T cells and PBMC, suggesting that Sanqimycin E can selectively inhibit T cell activation and proliferation. Sanqimycin E does not inhibit the expression of T cell activation markers IL-2 and CD25, but blocks the activation of T cell cycle in G0/G1 phase, suggesting that the signal transduction phase of Sanqimycin E action may be located after T cell activation to cell cycle initiation. Sanqimycin E significantly inhibits the secretion of the inflammatory cytokines IL-6 and IL-17A by activated T cells. Sanqimycin E significantly inhibited IL-2 induced proliferation of activated T cel
	2019-11
文献类型	学位论文
条目标识符	http://ir.kib.ac.cn/handle/151853/74229
专题	昆明植物所硕博研究生毕业学位论文
推荐引用方式 GB/T 7714	周知. 微生物来源的天然产物的免疫抑制活性的筛选及评价, Screening and Evaluation of Microbial-derived Natural Products with Immunosuppressive Activity[D],2019.

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