CpG-ODN与受体TLR9在酸性条件下特异性结合的研究
发布时间：2018-10-18 17:25
【摘要】：CpG-ODN是一些发现于细菌中含有未甲基化的CpG二核苷酸对并且具有免疫激活作用的寡核苷酸。大量研究已证明CpG-ODN具有直接激活B细胞增殖、促进树突状细胞成熟及诱导抗原呈递细胞分泌多种细胞因子等作用。应用基础方面的研究表明CpG-ODN可以作为一类新型的免疫佐剂在抗肿瘤、抗感染和防治哮喘等方面具有非常乐观的应用前景。鉴于CpG-ODN广泛的免疫调节作用及应用潜力,对其细胞内分子作用机制的研究成为当前的一个热点,而焦点问题是寻找免疫细胞上CpG-ODN的特异性识别受体并阐明其信号跨膜传递的机制。 最近的研究发现CpG-ODN的特异性识别受体极可能是Toll-Like-Receptors(TLRs)家族的新成员——TLR9,证据主要来自基因敲除小鼠,即如果将TLR9或TLR家族共用的下游信号分子的接头分子——MyD88的基因敲除后,则基因敲除小鼠的免疫细胞就失去了对CpG-ODN刺激的应答性,而且TLR9是一种膜蛋白,其胞外部分含有与甲基化CpG-DNA结合蛋白MBD-1-4同源的DNA结合域,这提示CpG-ODN系与TLR9结合之后再通过接头分子MyD88向内传递激活信号。但令人困惑的是目前一直未能直接观察到TLR9与CpG-ODN的特异性相互作用,而且免疫细胞膜上并不存在CpG-ODN的特异性结合位点。尽管ODN能与细胞膜结合,但这些结合均没有CpG序列的特异性,而且证据表明免疫细胞对CpG-ODN的特异性识别及信号触发不是发生于细胞外膜上,而是继发于CpG-ODN被细胞内吞和内体酸化之后。据此我们推测:如果TLR9的确是CpG-ODN的特异性受体,则TLR9与CpG-ODN的特异性结合及相关激活信号的触发极有可能发生于CpG-ODN被内吞之后的酸性内体中,而不是发生于细胞外膜上。亦即TLR9与CpG-ODN的特异性结合极有可能需要一个特定的外在条件即偏酸性的pH环境。 为证实上述推测,我们在研究中比较了不同pH条件下CpG-ODN与细胞膜的结合及其免疫调节活性,发现:在中性pH条件下ODNs与免疫细胞膜的结合的确没有序列选择性,但在偏酸性pH条件却能观察到CpG-ODN与免疫细胞膜有特异性结合,而且在微偏酸条件下,免疫细胞对CpG-ODN的刺激更加敏感。另外,我们以RT-PCR方法从小鼠脾细胞mRNA中扩增出TLR9基因,将胞外区亚克隆至pcDNA3.1真核表达载体,转入COS7细胞瞬时表达,表达产物可被抗小鼠TLR9
[Abstract]:CpG-ODN is a group of oligonucleotides found in bacteria that contain unmethylated CpG dinucleotides and are immune-activated. A large number of studies have proved that CpG-ODN can directly activate the proliferation of B cells, promote the maturation of dendritic cells and induce the secretion of many cytokines by antigen-presenting cells. It is shown that CpG-ODN can be used as a new kind of immune adjuvant in anti-tumor, anti-infection and asthma prevention and treatment. In view of the wide range of immunomodulatory effects and potential applications of CpG-ODN, the study of its intracellular molecular mechanism has become a hot topic. The focus is to identify the specific receptors of CpG-ODN on immune cells and to elucidate the mechanism of its signal transmembrane transmission. Recent studies have found that the specific recognition receptor for CpG-ODN is likely to be a new member of the Toll-Like-Receptors (TLRs) family-TLR9, evidence comes mainly from gene knockout mice, that is, if the MyD88 gene, the joint molecule of downstream signaling molecules shared by the TLR9 or TLR family, is knocked out, The immune cells of the knockout mice lose their responsiveness to CpG-ODN stimulation, and TLR9 is a membrane protein that contains a DNA binding domain homologous to the methylated CpG-DNA binding protein MBD-1-4. This suggests that the CpG-ODN system binds to TLR9 and then transmits the activation signal inward through the junction molecule MyD88. However, it is puzzling that the specific interaction between TLR9 and CpG-ODN has not been observed directly, and there is no specific binding site of CpG-ODN on the immune cell membrane. Although ODN binds to the cell membrane, none of these bindings are specific to CpG sequences, and the evidence suggests that the specific recognition and signal triggering of CpG-ODN by immune cells do not occur on the extracellular membrane. It occurs after CpG-ODN is endocytosis and endosome acidification. We speculate that if TLR9 is indeed a specific receptor of CpG-ODN, the specific binding of TLR9 to CpG-ODN and the trigger of related activation signals are likely to occur in the acidic endosome after the endocytosis of CpG-ODN, rather than on the extracellular membrane. In other words, the specific binding of TLR9 to CpG-ODN is likely to require a specific external condition, that is, a slightly acidic pH environment. In order to confirm the above conjecture, we compared the binding and immunomodulatory activity of CpG-ODN to cell membrane under different pH conditions. It was found that the binding of ODNs to immune cell membrane under neutral pH did not have sequence selectivity. However, the specific binding of CpG-ODN to the immune cell membrane was observed under the condition of slightly acidic pH, and the immune cells were more sensitive to the stimulation of CpG-ODN under the condition of slight acid. In addition, we amplified the TLR9 gene from mouse spleen cell mRNA by RT-PCR method, subcloned the extracellular region into pcDNA3.1 eukaryotic expression vector and transferred it into COS7 cell for transient expression. The expression product can be expressed by anti-mouse TLR9.
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R392
，
本文编号：2279858
[Abstract]:CpG-ODN is a group of oligonucleotides found in bacteria that contain unmethylated CpG dinucleotides and are immune-activated. A large number of studies have proved that CpG-ODN can directly activate the proliferation of B cells, promote the maturation of dendritic cells and induce the secretion of many cytokines by antigen-presenting cells. It is shown that CpG-ODN can be used as a new kind of immune adjuvant in anti-tumor, anti-infection and asthma prevention and treatment. In view of the wide range of immunomodulatory effects and potential applications of CpG-ODN, the study of its intracellular molecular mechanism has become a hot topic. The focus is to identify the specific receptors of CpG-ODN on immune cells and to elucidate the mechanism of its signal transmembrane transmission. Recent studies have found that the specific recognition receptor for CpG-ODN is likely to be a new member of the Toll-Like-Receptors (TLRs) family-TLR9, evidence comes mainly from gene knockout mice, that is, if the MyD88 gene, the joint molecule of downstream signaling molecules shared by the TLR9 or TLR family, is knocked out, The immune cells of the knockout mice lose their responsiveness to CpG-ODN stimulation, and TLR9 is a membrane protein that contains a DNA binding domain homologous to the methylated CpG-DNA binding protein MBD-1-4. This suggests that the CpG-ODN system binds to TLR9 and then transmits the activation signal inward through the junction molecule MyD88. However, it is puzzling that the specific interaction between TLR9 and CpG-ODN has not been observed directly, and there is no specific binding site of CpG-ODN on the immune cell membrane. Although ODN binds to the cell membrane, none of these bindings are specific to CpG sequences, and the evidence suggests that the specific recognition and signal triggering of CpG-ODN by immune cells do not occur on the extracellular membrane. It occurs after CpG-ODN is endocytosis and endosome acidification. We speculate that if TLR9 is indeed a specific receptor of CpG-ODN, the specific binding of TLR9 to CpG-ODN and the trigger of related activation signals are likely to occur in the acidic endosome after the endocytosis of CpG-ODN, rather than on the extracellular membrane. In other words, the specific binding of TLR9 to CpG-ODN is likely to require a specific external condition, that is, a slightly acidic pH environment. In order to confirm the above conjecture, we compared the binding and immunomodulatory activity of CpG-ODN to cell membrane under different pH conditions. It was found that the binding of ODNs to immune cell membrane under neutral pH did not have sequence selectivity. However, the specific binding of CpG-ODN to the immune cell membrane was observed under the condition of slightly acidic pH, and the immune cells were more sensitive to the stimulation of CpG-ODN under the condition of slight acid. In addition, we amplified the TLR9 gene from mouse spleen cell mRNA by RT-PCR method, subcloned the extracellular region into pcDNA3.1 eukaryotic expression vector and transferred it into COS7 cell for transient expression. The expression product can be expressed by anti-mouse TLR9.
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R392
，
本文编号：2279858
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