【摘要】：CpG-ODN是一些發(fā)現(xiàn)于細(xì)菌中含有未甲基化的CpG二核苷酸對(duì)并且具有免疫激活作用的寡核苷酸。大量研究已證明CpG-ODN具有直接激活B細(xì)胞增殖、促進(jìn)樹突狀細(xì)胞成熟及誘導(dǎo)抗原呈遞細(xì)胞分泌多種細(xì)胞因子等作用。應(yīng)用基礎(chǔ)方面的研究表明CpG-ODN可以作為一類新型的免疫佐劑在抗腫瘤、抗感染和防治哮喘等方面具有非常樂(lè)觀的應(yīng)用前景。鑒于CpG-ODN廣泛的免疫調(diào)節(jié)作用及應(yīng)用潛力,對(duì)其細(xì)胞內(nèi)分子作用機(jī)制的研究成為當(dāng)前的一個(gè)熱點(diǎn),而焦點(diǎn)問(wèn)題是尋找免疫細(xì)胞上CpG-ODN的特異性識(shí)別受體并闡明其信號(hào)跨膜傳遞的機(jī)制。 最近的研究發(fā)現(xiàn)CpG-ODN的特異性識(shí)別受體極可能是Toll-Like-Receptors(TLRs)家族的新成員——TLR9,證據(jù)主要來(lái)自基因敲除小鼠,即如果將TLR9或TLR家族共用的下游信號(hào)分子的接頭分子——MyD88的基因敲除后,則基因敲除小鼠的免疫細(xì)胞就失去了對(duì)CpG-ODN刺激的應(yīng)答性,而且TLR9是一種膜蛋白,其胞外部分含有與甲基化CpG-DNA結(jié)合蛋白MBD-1-4同源的DNA結(jié)合域,這提示CpG-ODN系與TLR9結(jié)合之后再通過(guò)接頭分子MyD88向內(nèi)傳遞激活信號(hào)。但令人困惑的是目前一直未能直接觀察到TLR9與CpG-ODN的特異性相互作用,而且免疫細(xì)胞膜上并不存在CpG-ODN的特異性結(jié)合位點(diǎn)。盡管ODN能與細(xì)胞膜結(jié)合,但這些結(jié)合均沒(méi)有CpG序列的特異性,而且證據(jù)表明免疫細(xì)胞對(duì)CpG-ODN的特異性識(shí)別及信號(hào)觸發(fā)不是發(fā)生于細(xì)胞外膜上,而是繼發(fā)于CpG-ODN被細(xì)胞內(nèi)吞和內(nèi)體酸化之后。據(jù)此我們推測(cè):如果TLR9的確是CpG-ODN的特異性受體,則TLR9與CpG-ODN的特異性結(jié)合及相關(guān)激活信號(hào)的觸發(fā)極有可能發(fā)生于CpG-ODN被內(nèi)吞之后的酸性內(nèi)體中,而不是發(fā)生于細(xì)胞外膜上。亦即TLR9與CpG-ODN的特異性結(jié)合極有可能需要一個(gè)特定的外在條件即偏酸性的pH環(huán)境。 為證實(shí)上述推測(cè),我們?cè)谘芯恐斜容^了不同pH條件下CpG-ODN與細(xì)胞膜的結(jié)合及其免疫調(diào)節(jié)活性,發(fā)現(xiàn):在中性pH條件下ODNs與免疫細(xì)胞膜的結(jié)合的確沒(méi)有序列選擇性,但在偏酸性pH條件卻能觀察到CpG-ODN與免疫細(xì)胞膜有特異性結(jié)合,而且在微偏酸條件下,免疫細(xì)胞對(duì)CpG-ODN的刺激更加敏感。另外,我們以RT-PCR方法從小鼠脾細(xì)胞mRNA中擴(kuò)增出TLR9基因,將胞外區(qū)亞克隆至pcDNA3.1真核表達(dá)載體,轉(zhuǎn)入COS7細(xì)胞瞬時(shí)表達(dá),表達(dá)產(chǎn)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.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2006
【分類號(hào)】：R392
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本文編號(hào)：2279858