小鼠树突状细胞TLR7表达及其介导的免疫应答初步研究

发布时间：2018-03-11 18:44

本文选题：DC2.4　切入点：Toll样受体7　出处：《第四军医大学》2008年硕士论文　论文类型：学位论文

【摘要】： 树突状细胞(Dendritic cell, DC)作为体内功能最强的专职抗原提呈细胞,主要依赖其表面的Toll样受体(Toll like receptors,TLRs)识别病原微生物,诱导固有免疫(innate immunity)应答,激活特异性免疫应答。Toll样受体7(Toll like receptor 7,TLR7)是近年发现的一类特殊的TLRs,广泛存在于体内免疫细胞中,尤以DC为著。业已证实,TLR7主要作为某些小分子抗病毒化合物和病毒单链RNA(single strand RNA,ssRNA)分子的模式识别受体(pattern recognition receptors,PRR),介导DC发挥免疫应答效应。迄今为止,人们对TLR7的亚细胞定位及其识别配体后介导的免疫效应尚不完全清楚。因此,对TLR7的深入研究,将进一步揭示机体抗感染免疫的分子机制。为了解DC中TLR7介导的免疫反应,本课题采用小鼠永生化的树突状细胞系DC2.4为细胞模型,检测了DC2.4中TLR7和TLR4的表达及TLR7识别配体Imiquimod后介导的DC免疫应答效应。本课题的研究内容和实验结果如下: 1. DC2.4中TLR7基因和蛋白表达检测根据Gene bank中的小鼠TLR7和β-actin基因全序列,分别设计相应上、下游引物各一对,用反转录PCR(RT-PCR)检测DC2.4中TLR7 mRNA表达,Western blot检测DC2.4中TLR7蛋白表达。结果显示,DC2.4中TLR7只有mRNA转录,检测不到蛋白表达。 2. DC2.4中TLR4蛋白表达检测在放有盖玻片的六孔板中,用含10%胎牛血清的RPMI 1640培养基培养DC2.4,待盖玻片上细胞长至约50～60%时,用间接免疫荧光法检测DC2.4中TLR4蛋白表达。结果显示,DC2.4细胞膜表面可检测到TLR4蛋白表达。 3. TLR4的配体——LPS刺激DC2.4,诱导TLR7蛋白的表达培养DC2.4至对数生长期,培养液中加入LPS(1 mg/L),分别在12 h、24 h、48 h、72 h收集细胞,提取蛋白,以未加入LPS组为阴性对照,胎盘组织提取蛋白为阳性对照,用Western blot检测蛋白表达情况。结果表明,LPS刺激DC2.4后12 h,可检测到TLR7蛋白的表达,但72 h后未能检测到。阴性对照组无TLR7蛋白表达,阳性对照组有明显的TLR7蛋白表达。 4. TLR7识别配体Imiquimod介导的DC免疫应答效应将DC2.4按1×108 /L接种于96孔板,共分4组,每组均设复孔。设L0组为阴性对照组,L组加入LPS(1 mg/L),I组加入抗病毒化合物Imiquimod(10 mol/L),LI组先加入LPS(1 mg/L),12h后加入Imiquimod(10 mol/L)。培养12h后收获各组细胞培养上清,离心后用ELISA方法检测IL-12和IFN-α表达水平,操作严格按照试剂盒说明书进行。数据采用SPSS11.0软件进行统计处理。结果显示,Imiquimod刺激表达TLR7蛋白的DC2.4后,IL-12分泌显著增加,IFN-α分泌无显著变化。结论: 1.在DC2.4中有TLR4蛋白和TLR7 mRNA表达,但检测不到TLR7蛋白表达。 2.TLR4天然配体LPS刺激12h后,可诱导DC2.4表达TLR7蛋白,但72h后未能检测到蛋白表达。 3.在DC2.4中,诱导表达的TLR7蛋白分子可以识别配体Imiquimod,激活信号转导途径,介导DC分泌免疫因子,IL-12分泌显著增加,而IFN-α分泌无显著变化。
[Abstract]:Dendritic cells (DCs), as the most functional professional antigen presenting cells in vivo, mainly rely on the surface Toll like receptor Toll like receptor TLRs) to recognize pathogenic microorganisms and induce innate immune response. Activation of specific immune response. Toll-like receptor 7 Toll like receptor 7 (TLR7) is a special class of TLRswhich is widely found in immune cells in vivo. Especially DC. It has been proved that TLR7 is mainly used as a pattern recognition receptor for some small molecule antiviral compounds and viral single-stranded RNA(single strand RNAs, which mediates the immune response effect of DC. The subcellular localization of TLR7 and the immune effect mediated by its ligand recognition have not been fully understood. Therefore, the further study of TLR7 will further reveal the molecular mechanism of anti-infective immunity. In order to understand the immune response mediated by TLR7 in DC, a murine immortalized dendritic cell line DC2.4 was used as a cell model to detect the expression of TLR7 and TLR4 in DC2.4 and the immune response of DC mediated by TLR7 recognition ligand Imiquimod. The research contents and experimental results are as follows:. 1. Detection of TLR7 gene and protein expression in DC2.4. According to the whole sequence of mouse TLR7 and 尾 -actin gene in Gene bank, a pair of upstream and downstream primers were designed to detect the expression of TLR7 mRNA in DC2.4 and TLR7 protein in DC2.4 by reverse transcription PCR RT-PCR. The results showed that only TLR7 mRNA was transcribed in DC2.4. Protein expression was not detected. 2. Detection of TLR4 protein expression in DC2.4. DC2.4 was cultured in a six-hole plate containing 10% fetal bovine serum in a six-hole plate containing 10% fetal bovine serum. When the cells on the cover glass reached about 50 ~ 60 cm, Indirect immunofluorescence assay was used to detect the expression of TLR4 protein in DC2.4. The results showed that the expression of TLR4 protein could be detected on the surface of DC2.4 cell membrane. 3. Ligands of TLR4 stimulate DC2.4 and induce the expression of TLR7 protein. When DC2.4 was cultured to logarithmic growth stage, LPS(1 mg / L was added to the culture medium, and cells were collected at 12 h, 24 h, 48 h and 72 h, respectively. The protein was extracted from the cells from the control group without LPS, and the positive control was from the placental tissue. Western blot was used to detect the expression of TLR7 protein. The results showed that the expression of TLR7 protein could be detected 12 h after DC2.4 stimulation, but not at 72 h. There was no expression of TLR7 protein in the negative control group, but there was obvious TLR7 protein expression in the positive control group. 4. Effect of TLR7 recognition ligand Imiquimod mediated DC immune response. DC2.4 was inoculated on 96 well plate according to 1 脳 10 ~ 8 / L, and divided into 4 groups, each group was divided into four groups, and each group was divided into four groups: L0 group as negative control group, L group as negative control group, adding LPS(1 mg / L Li group, adding antiviral compound Imiquimod(10 mol / L group for 12 h, then adding Imiquimod(10 mol / L group. After 12 h culture, the supernatant of each group was harvested, and the cell culture supernatant of each group was harvested after 12 h culture. After centrifugation, the expression levels of IL-12 and IFN- 伪 were detected by ELISA method, and the data were analyzed by SPSS11.0 software. The results showed that the IL-12 secretion of DC2.4 stimulated by Imiquimod had no significant change. Conclusion:. 1. TLR4 and TLR7 mRNA were expressed in DC2.4, but TLR7 protein was not detected. 2. TLR4 natural ligand LPS could induce the expression of TLR7 protein in DC2.4 for 12 h, but the expression of TLR7 protein could not be detected after 72 h. 3. In DC2.4, the induced expression of TLR7 protein could recognize the ligand Imiquimod, activate the signal transduction pathway, and mediate the secretion of IL-12 by DC, but the secretion of IFN- 伪 did not change significantly.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392

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