一种人微卫星DNA来源的抑制性寡核苷酸功能及构效研究

发布时间：2019-04-21 21:04

【摘要】：固有免疫系统是机体抵抗病原体入侵的第一道防线。天然免疫细胞通过其上的模式识别受体（PRR）识别微生物的病原体相关分子模式（PAMP），进而经由信号的级联放大诱导I型干扰素和前炎症细胞因子的产生来清除入侵的病原体。Toll样受体（TLR）是目前研究最多的一类PRR家族。在正常的生理条件下，TLR介导的固有免疫的激活有助于机体防御病原体的侵袭。但如果这种激活得不到有效的控制，固有免疫系统会被过度激活并产生过量的细胞因子，，导致炎症和自身免疫性疾病。为了治疗这些疾病，发展抑制固有免疫过度激活的药物是十分必要的。在本室前期工作中，我们发现一种微卫星DNA来源的抑制性寡核苷酸（ODN）SAT05f（5′-CCTCCTCCTCCTCCTCCTCCTCCT-3′）可以抑制病毒核酸诱导的人PBMC产生IFN-α、保护小鼠抵抗D-GalN/CpG ODN导致的致死性休克以及减少慢性移植物抗宿主病狼疮样小鼠体内抗双链DNA抗体的产生。这些研究结果都展示了SAT05f用于治疗过度免疫激活介导疾病的良好潜力。在本研究中，我们尝试发掘更多的SAT05f的免疫抑制功能并研究SAT05f的构效关系。通过实验发现SAT05f可以抑制CpG ODN诱导的小鼠脾细胞中B淋巴细胞的增殖、抑制小鼠脾细胞中B淋巴细胞结合和摄入CpG ODN、抑制CpG ODN诱导的RAW264.7细胞产生TNF-α、抑制CpG ODN诱导的RAW264.7细胞中TLR9mRNA表达的上调，以及抑制CpG ODN诱导的类pDC细胞（CAL-1）的激活。此外，250μg剂量的SAT05f可以增加热灭活菌诱导的脓毒性休克小鼠的生存率。构效研究显示：1）CCT重复ODN至少要含有8个CCT单元才能发挥抑制活性。2）SAT05f的3′末端CCT单元对于其抑制活性的发挥是必要的。3）SAT05f的5′末端CCT单元可以被优化而获得活性更强的抑制ODN。这些结果将有助于发展一种治疗TLR过度激活介导疾病的抑制性寡核苷酸药物。
[Abstract]:The innate immune system is the first line of defense against pathogen invasion. Innate immune cells recognize the pathogen-related molecular pattern (PAMP), of microorganisms through the pattern recognition receptor (PRR) on it Toll-like receptor (TLR) (Toll-like receptor) is the most widely studied type of PRR family to eliminate invasive pathogens by inducing the production of interferon I and proinflammatory cytokines by cascade amplification of signals. Under normal physiological conditions, the activation of innate immunity mediated by TLR contributes to the defense against pathogen invasion. But if this activation is not effectively controlled, the innate immune system will be over-activated and produce excessive cytokines, leading to inflammation and autoimmune diseases. In order to treat these diseases, it is necessary to develop drugs that inhibit intrinsic immune hyperactivation. In our previous work, we found that a microsatellite DNA-derived inhibitory oligodeoxynucleotides (ODN) SAT05f (5-CCTCCTCCTCCTCCTCCTCCTCCTCCTCCTCCTCCTCCTCCTCCTCCTCCTCCTCCTCCTCCTCCTCCTCCTCCTCCTCCTCCTCCTCCTCCTCCTCCTCCT Protect mice against D-GalN/CpG ODN-induced fatal shock and reduce the production of anti-double-stranded DNA antibodies in lupus-like mice with chronic graft-versus-host disease. These findings demonstrate the good potential of SAT05f in the treatment of diseases mediated by excessive immune activation. In this study, we tried to explore more immunosuppressive functions of SAT05f and study the structure-activity relationship of SAT05f. It was found that SAT05f could inhibit the proliferation of B lymphocytes in mouse spleen cells induced by CpG ODN, inhibit the binding of B lymphocytes to spleen cells in mice and inhibit the production of TNF- 伪 by RAW264.7 cells induced by CpG ODN. It also inhibited the up-regulation of TLR9mRNA expression in RAW264.7 cells induced by CpG ODN and the activation of pDC-like cells (CAL-1) induced by CpG ODN. In addition, 250 渭 g of SAT05f increased the survival rate of septic shock mice induced by thermally inactivated bacteria. Structure-activity studies show that: 1) CCT repeat ODN must contain at least eight CCT units in order to exert inhibitory activity. 2) the 3 'terminal CCT unit of SAT05f is necessary for its inhibitory activity. 3) the 5' terminal CCT unit of SAT05f can perform its inhibitory activity. To obtain a more active inhibition of ODN. by being optimized These results will contribute to the development of an inhibitory oligodeoxynucleotide drug for the treatment of TLR over-activation-mediated diseases.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R341

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