本文選題：DC2.4　切入點：Toll樣受體7　出處：《第四軍醫(yī)大學(xué)》2008年碩士論文　論文類型：學(xué)位論文

【摘要】： 樹突狀細(xì)胞(Dendritic cell, DC)作為體內(nèi)功能最強的專職抗原提呈細(xì)胞,主要依賴其表面的Toll樣受體(Toll like receptors,TLRs)識別病原微生物,誘導(dǎo)固有免疫(innate immunity)應(yīng)答,激活特異性免疫應(yīng)答。Toll樣受體7(Toll like receptor 7,TLR7)是近年發(fā)現(xiàn)的一類特殊的TLRs,廣泛存在于體內(nèi)免疫細(xì)胞中,尤以DC為著。業(yè)已證實,TLR7主要作為某些小分子抗病毒化合物和病毒單鏈RNA(single strand RNA,ssRNA)分子的模式識別受體(pattern recognition receptors,PRR),介導(dǎo)DC發(fā)揮免疫應(yīng)答效應(yīng)。迄今為止,人們對TLR7的亞細(xì)胞定位及其識別配體后介導(dǎo)的免疫效應(yīng)尚不完全清楚。因此,對TLR7的深入研究,將進一步揭示機體抗感染免疫的分子機制。 為了解DC中TLR7介導(dǎo)的免疫反應(yīng),本課題采用小鼠永生化的樹突狀細(xì)胞系DC2.4為細(xì)胞模型,檢測了DC2.4中TLR7和TLR4的表達及TLR7識別配體Imiquimod后介導(dǎo)的DC免疫應(yīng)答效應(yīng)。 本課題的研究內(nèi)容和實驗結(jié)果如下: 1. DC2.4中TLR7基因和蛋白表達檢測 根據(jù)Gene bank中的小鼠TLR7和β-actin基因全序列,分別設(shè)計相應(yīng)上、下游引物各一對,用反轉(zhuǎn)錄PCR(RT-PCR)檢測DC2.4中TLR7 mRNA表達,Western blot檢測DC2.4中TLR7蛋白表達。結(jié)果顯示,DC2.4中TLR7只有mRNA轉(zhuǎn)錄,檢測不到蛋白表達。 2. DC2.4中TLR4蛋白表達檢測 在放有蓋玻片的六孔板中,用含10%胎牛血清的RPMI 1640培養(yǎng)基培養(yǎng)DC2.4,待蓋玻片上細(xì)胞長至約50～60%時,用間接免疫熒光法檢測DC2.4中TLR4蛋白表達。結(jié)果顯示,DC2.4細(xì)胞膜表面可檢測到TLR4蛋白表達。 3. TLR4的配體——LPS刺激DC2.4,誘導(dǎo)TLR7蛋白的表達 培養(yǎng)DC2.4至對數(shù)生長期,培養(yǎng)液中加入LPS(1 mg/L),分別在12 h、24 h、48 h、72 h收集細(xì)胞,提取蛋白,以未加入LPS組為陰性對照,胎盤組織提取蛋白為陽性對照,用Western blot檢測蛋白表達情況。結(jié)果表明,LPS刺激DC2.4后12 h,可檢測到TLR7蛋白的表達,但72 h后未能檢測到。陰性對照組無TLR7蛋白表達,陽性對照組有明顯的TLR7蛋白表達。 4. TLR7識別配體Imiquimod介導(dǎo)的DC免疫應(yīng)答效應(yīng) 將DC2.4按1×108 /L接種于96孔板,共分4組,每組均設(shè)復(fù)孔。設(shè)L0組為陰性對照組,L組加入LPS(1 mg/L),I組加入抗病毒化合物Imiquimod(10 mol/L),LI組先加入LPS(1 mg/L),12h后加入Imiquimod(10 mol/L)。培養(yǎng)12h后收獲各組細(xì)胞培養(yǎng)上清,離心后用ELISA方法檢測IL-12和IFN-α表達水平,操作嚴(yán)格按照試劑盒說明書進行。數(shù)據(jù)采用SPSS11.0軟件進行統(tǒng)計處理。結(jié)果顯示,Imiquimod刺激表達TLR7蛋白的DC2.4后,IL-12分泌顯著增加,IFN-α分泌無顯著變化。 結(jié)論: 1.在DC2.4中有TLR4蛋白和TLR7 mRNA表達,但檢測不到TLR7蛋白表達。 2.TLR4天然配體LPS刺激12h后,可誘導(dǎo)DC2.4表達TLR7蛋白,但72h后未能檢測到蛋白表達。 3.在DC2.4中,誘導(dǎo)表達的TLR7蛋白分子可以識別配體Imiquimod,激活信號轉(zhuǎn)導(dǎo)途徑,介導(dǎo)DC分泌免疫因子,IL-12分泌顯著增加,而IFN-α分泌無顯著變化。
[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- 偽 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- 偽 did not change significantly.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R392

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