TLR配体诱导骨髓来源树突状细胞获得产生全反式维甲酸的能力

发布时间：2018-01-20 12:48

本文关键词： 树突状细胞维甲酸 Toll样受体 IgA 肠黏膜归巢　出处：《浙江大学》2011年硕士论文　论文类型：学位论文

【摘要】：背景和目的粘膜是机体抵抗病原微生物感染的第一道防线,目前发现90%以上的人类传染病通过粘膜途径感染。粘膜免疫在机体免疫系统中占有重要的地位,它能够抵抗致病菌的入侵,对饮食和呼吸中的有害抗原,作出适当的免疫应答,同时对环境和食物中的无害抗原及共生菌,表现为免疫耐受。所以,如果能够控制粘膜感染,就可控制大部分传染病的发生。人体肠粘膜总面积为300m2,是机体内外环境交流的主要界面,是粘膜感染过程中的易发部位。肠道粘膜免疫系统由肠相关淋巴组织(gut-associated lymphoid tissue, GALT)组成,是机体免疫系统中最大最复杂的网络。具有严格的免疫反应机制和完善的免疫调控机制。派氏结(PP)、肠系膜淋巴结(MLN)及孤立淋巴滤泡是肠道黏膜免疫应答的诱导部位,负责起始肠道内的免疫应答；肠道黏膜上皮内淋巴细胞(IEL)及固有层(LP)内散在的淋巴细胞是肠道黏膜免疫应答的效应细胞。与系统免疫应答相比,粘膜免疫系统的一个显著的特点是产生分泌型IgA(secretory IgA, sIgA)。人类肠道每天能够分泌3g以上的sIgA。已知IgA占体内总免疫球蛋白75%以上,其中大部分是位于粘膜表面的sIgA。sIgA对保持肠道上皮的稳态起重要作用,它可与入侵的病原微生物结合,阻止其在粘膜上皮细胞表面的定植,并能中和致病菌产生的毒素和侵袭性酶类。此外,sIgA对由食物摄入和空气吸入的某些抗原物质具有免疫排斥作用,可封闭这些抗原,使其游离于粘膜表面不致进入机体,从而避免引起全身的免疫反应,减少局部过敏反应的发生。树突状细胞(DC)是功能最强的抗原提呈细胞,是固有免疫和适应性免疫的桥梁和纽带,其调节机体免疫应答的类型和强度。业已发现肠道DC有与脾脏、胸腺、腋窝淋巴结和腹股沟淋巴结来源的DC不同的特性,前者能产生RA,而后者不产生RA。肠黏膜DC产生的RA是粘膜免疫应答的一个关键性调节因素,RA能诱导T、B淋巴细胞表达肠黏膜归巢受体,从而赋予在肠相关淋巴组织中活化的T、B淋巴细胞经过淋巴细胞再循环后,重新归巢到肠黏膜的特性；RA参与B细胞IgA类别转换；RA还能影响肠黏膜Treg和Th17的分化,从而可能在炎症性肠病的发生和发展中发挥作用。但是肠黏膜DC产生RA的机制——即什么信号启动DC产生RA迄今尚未阐明。晚近有学者发现,皮肤和肺中的DC也能产生RA。值得注意的是：能产生RA的DC的组织来源与不产生RA的DC的组织来源相比,前者都与外界环境相通,存在大量的共生菌,而后者都处于无菌状态。所以我们推测,微生物的某些组成成分可能是DC产生RA的始动因素。本实验研究Toll样受体4和9(TLR4/9)的配体——LPS及CpG对骨髓来源DC合成RA能力的影响。方法 1.DC细胞分组处理：培养的骨髓细胞系——DC2.4经PBS洗涤后重悬,以1×106cells/well接种于24孔板,经下列三种不同处理,在37℃、5%CO2条件下培养48h。 1.1 LPS对DC产生RA影响的分组：①空白对照组,即DC组；②DC+LPS(10μg/ml)；③DC+LPS(1μg/ml);④DC+LPS(0.1μg/ml);⑤DC+LPS (0.01μg/ml);⑥DC+LPS(0.001μg/ml)。 1.2 CpG对DC产生RA影响的分组：①空白对照组,即DC组；②DC+CpG(10μM);③DC+CpG(1μM);④DC+CpG(0.1μM);⑤DC+CpG(0.01μM);⑥DC+ CpG(0.001μM)。 1.3 NF-KB阻断剂SN50对DC产生RA影响的分组：①空白对照组,即DC组;②DC+LPS(1μg/ml);③DC+LPS(1μg/ml)+SN50(18μM);④DC+CpG(1μM);⑤DC+CpG(1μM)+SN50(18μM)。 2.实时荧光定量PCR测定处理后DC的RA代谢相关酶(ALDH1a-1,-2,-3及ADH-1,-4,-5)的表达。 3.LPS或CpG处理细胞30和60分钟后,提取细胞浆和细胞核蛋白,Western blotting检测NF-KB蛋白表达。在之前培养体系中加入NF-κB蛋白阻断剂SN50,Western blotting检测阻断效应。 4.DC和B细胞共培养：采用流式细胞术分选C57BL/6小鼠脾脏B细胞(5×105),与1μg/ml的LPS或1μM的CpG预处理48h的DC(1×105)共培养4天。以10种方式处理细胞(IL-5和IL-6浓度10ng/ml)：空白对照组,即B细胞组；B+DC2.4细胞组；B+LPS-DC组;B+LPS-DC+IL-5组;B+LPS-DC+IL-6组;B+LPS-DC+IL-5+IL-6组；B+CpG-DC组；B+CpG-DC+IL-5组;B+CpG-DC+IL-6组;B+CpG-DC+IL-5+IL-6组。 5.收集共培养4天的细胞,流式细胞仪分析B细胞归巢受体α4β7和CCR9表达。 6.B细胞趋化试验：收集与DC共培养后的B细胞,Transwell细胞迁移试验检测B细胞对趋化因子MadCAM-1和TECK的趋化活性。 7.收集共培养4天的细胞培养上清,ELISA法检测上清中IgA浓度。 8. ELISA检测不同浓度LPS或CpG处理48h的DC培养上清中IL-6和TGF-β1的浓度。 9. Griess法检测不同浓度LPS或CpG处理48h的DC培养上清中NO浓度。结果 1.LPS或CpG能诱导骨髓来源的DC表达Aldh1a2 mRNA。1μg/ml LPS处理DC48小时后,Aldh1a2 mRNA表达增高最为明显,增高近10倍,ADH4表达上调增高18倍。1μM CpG处理DC后Aldh1a2 mRNA表达提高12.45倍,Aldh1a1 mRNA表达有近10倍的提高,ADH4表达上调近12倍。但无论任一浓度的LPS或CpG处理后,DC Aldh1a3 mRNA都没有发现明显的改变。 2.NF-κB信号通路在LPS或CpG诱导DC产生RA中发挥重要作用,用抑制剂SN50阻断后,NF-κB核转位被抑制,DC表达Aldh1a2 mRNA下降。SN50加入到LPS-DC (CpG-DC)体系30min后,NF-κB核转位即可被抑制。细胞核内NF-κB/Histone H3比由0.34(0.33)仅上升为0.46(0.48)；60min后阻断效应更加显著,核内NF-κB/Histone H3比0.44(0.47)。SN50使TLR-DC Aldhla2相对表达量增高幅度分别由10倍之多降为仅2倍。 3.LPS-或CpG-DC增强B细胞肠黏膜归巢受体α4β7和CCR9的表达,其中上调CCR9较α4β7明显,CCR9平均荧光强度均提高了1倍之多。IL-5和/或IL-6增强α4β7表达,但不影响CCR9表达。 4.LPS-或CpG-DC能增强B细胞对TECK的趋化活性,与对照组相比,分别提高了6.4和13.1个百分点。但LPS-或CpG-DC对B细胞对MadCAM-1的趋化活性影响较弱,仅分别提高2.5和6.25个百分点。 5.LPS-或CpG-DC促进B细胞分泌IgA,培养上清中IgA浓度与对照组相比分别提高近4倍和6倍。但外加的IL-5和/或IL-6并没有进一步促进IgA的分泌。 6.LPS或CpG增强DC产生IL-6,TGF-β和NO的能力。刺激物浓度在0.1-10区间内与细胞因子分泌成正相关,呈现剂量依赖性。10μg/ml LPS或10μM CpG刺激DC2.4后,其IL-6分泌量分别提高了1.3和1.5倍,TGF-β1分泌量分别增加了61%和70%。在10μg/ml LPS及10μM CpG时,NO分泌量达到最高,与对照组相比,分别提高了47%和84%。结论 TLR配体能触发骨髓来源DC获得产生RA的能力,提示肠道微生物在DC产生RA中发挥关键作用。
[Abstract]:Background and purpose
Mucosal is the body's first line of defense against infection of pathogenic microorganisms, the discovery of human infectious diseases more than 90% of the way through the mucous membrane infection. Mucosal immunity plays an important role in the immune system, which can resist the invasion of pathogenic bacteria, harmful antigen eating and breathing in, make appropriate immune responses, and harmless to the antigen the environment and food and the symbiotic bacteria showed immune tolerance. So, if we can control the mucosal infections can occur most infectious diseases.
Human intestinal mucosa of the total area of 300m2, is the main interface of internal and external environment of the body exchange, is susceptible of mucosal infections. In the process of intestinal mucosal immune system by the gut associated lymphoid tissue (gut-associated lymphoid, tissue, GALT), the immune system is the largest and most complex network. With strict and perfect the mechanism of immune response the immune regulation mechanism. Peyer's patch (PP), mesenteric lymph node (MLN) and isolated lymphoid follicles is part of mucosal immune response induced by the immune response in the intestine responsible for initiation; intestinal intraepithelial lymphocytes (IEL) and lamina propria (LP) in lymphocytes are scattered in the intestinal mucosal immune effector cells response. Compared with the systemic immune response, a remarkable feature of the mucosal immune system is the production of secretory IgA (secretory IgA sIgA). 3G can secrete more sIgA. known I human intestinal every day GA accounts for more than 75% in total immunoglobulin, most of which are located in the mucosal surface to maintain intestinal epithelial sIgA.sIgA homeostasis plays an important role, it can be combined with the invasion of pathogenic microorganisms, blocking its surface epithelial cell colonization, and enzymes and invasive toxin neutralizing pathogens. In addition, sIgA with immune rejection by some antigens and food intake air intake, can close the antigen, make it free from the mucosal surface does not enter the body, to avoid systemic immune response, reduce local allergic reactions.
Dendritic cells (DC) are the most potent antigen-presenting cells, is the bridge and link innate and adaptive immunity, the regulation of the immune response type and intensity. It has been found that intestinal DC with spleen, thymus, characteristics of axillary and inguinal lymph nodes from different DC, the former can produce RA. The latter does not produce RA. intestinal mucosa produced by DC RA is one of the key factors regulating the mucosal immune response, RA can induce T expression in intestinal mucosa, B lymphocyte homing receptor, thus giving activation in the gut associated lymphoid tissue T in B cells after lymphocyte recirculation after re homing to the characteristics of intestinal mucosa RA; B cells in IgA type conversion; RA can influence the differentiation of the intestinal mucosa of Treg and Th17, which may play a role in the occurrence and development of inflammatory bowel disease. But the intestinal mucosa DC RA mechanism -- that is what the letter No. DC RA has yet to start. In recent years some scholars have found that, skin and lung in DC can also produce RA. is worth noting: compared to RA can produce the DC source and does not produce RA DC tissue sources, the former are connected with the outside environment, there are a large number of symbiotic bacteria, while the latter are in the sterile condition. So we speculated that certain components of microorganisms may be the initial factor in DC RA. The experimental study of Toll like receptor 4 and 9 (TLR4/9) - LPS and CpG ligand effect on bone marrow derived DC RA synthesis ability.
Method
1.DC cell grouping treatment: the cultured bone marrow cell line DC2.4 was washed and washed after PBS, and was inoculated on 24 hole plates at 1 * 106cells/well, and then cultured under 37 different temperatures and 37 treatments at 37 5%CO2.
1.1 LPS RA group's effect on DC: blank control group, DC group; the DC+LPS (10 g/ml); the DC+LPS (1 g/ml); the DC+LPS (0.1 g/ml); the DC+LPS (0.01 g/ml); the DC+LPS (0.001 g/ml).
1.2 CpG RA group's effect on DC: blank control group, DC group; the DC+CpG (10 M); the DC+CpG (1 M); the DC+CpG (0.1 M); the DC+CpG (0.01 M); DC+ CpG (0.001 M).
1.3, the NF-KB blocker SN50 affects the production of RA in DC group: 1. The blank control group, namely DC group; DC+LPS (1 g/ml); DC+LPS (1 g/ml g/ml) +SN50 (18 18 M); (4) g/ml (1 mu); (5) (1)) (18).
The expression of RA metabolizing enzymes (ALDH1a-1, -2, -3, ADH-1, -4, -5) of DC after treatment was measured by 2. real time fluorescence quantitative PCR.
After treatment with 3.LPS or CpG for 30 and 60 minutes, the plasma and nuclear protein were extracted, and the expression of NF-KB protein was detected by Western blotting. NF- B B protein blocker SN50 and Western blotting were used to detect blocking effect in the previous culture system.
Co cultured 4.DC and B cells by flow cytometry C57BL/6 mouse spleen B cells (5 * 105), and 1 g/ml LPS or 1 M CpG 48h DC pretreatment (1 x 105) were cultured for 4 days. Cells were treated in 10 ways (IL-5 and IL-6 concentration of 10ng/ml): blank control group, B cells group; B+DC2.4 cell group; B+LPS-DC group; B+LPS-DC+IL-5 group; B+LPS-DC+IL-6 group; B+LPS-DC+IL-5+IL-6 group; B+CpG-DC group; B+CpG-DC+IL-5 group; B+CpG-DC+IL-6 group; B+CpG-DC+IL-5+IL-6 group.
5. the cells were cultured for 4 days, and the flow cytometry was used to analyze the expression of B cell homing receptor alpha 4 beta 7 and CCR9.
6.B cell chemotaxis test: B cells co cultured with DC were collected, and Transwell cell migration test was used to detect the chemotaxis activity of B cells to chemokine MadCAM-1 and TECK.
7. the cell culture supernatant was collected for 4 days, and the concentration of IgA in the supernatant was detected by ELISA method.
8. ELISA was used to detect the concentration of IL-6 and TGF- beta 1 in DC culture supernatant with different concentrations of LPS or CpG for 48h treatment.
9. Griess method was used to detect the concentration of NO in DC culture supernatant with different concentrations of LPS or CpG for 48h treatment.
Result
The expression of 1.LPS or CpG can induce bone marrow derived DC Aldh1a2 mRNA.1 g/ml LPS after DC48 hours of treatment, the expression of mRNA Aldh1a2 increased most obviously, increased nearly 10 times, ADH4 expression was 18 times.1 M CpG DC Aldh1a2 mRNA expression after treatment increased 12.45 times and Aldh1a1 mRNA expression were nearly 10 times increase, ADH4 expression of nearly 12 times. But regardless of any concentration of LPS or CpG treatment, DC Aldh1a3 mRNA found no obvious change.
2.NF- B signaling pathway in LPS induced by CpG or DC have played an important role in RA, blocked by inhibitors of SN50, NF- kappa B nuclear translocation was inhibited, the expression of DC Aldh1a2 decreased mRNA.SN50 into LPS-DC (CpG-DC) 30min system, NF- kappa B nuclear translocation can be inhibited. The nucleus of NF- kappa B/ Histone H3 ratio from 0.34 (0.33) only increased to 0.46 (0.48); 60min the blocking effect was more significant, nuclear NF- kappa B/Histone H3 0.44 (0.47).SN50 TLR-DC Aldhla2 relative expression increases by as much as 10 times reduced to 2 times.
3.LPS- or CpG-DC enhanced the expression of homing receptor alpha 4 beta 7 and CCR9 in B cells. The up regulation of CCR9 was significantly higher than that of the alpha 4 beta 7, and the mean fluorescence intensity of CCR9 increased by 1 times..IL-5 and / or IL-6 enhanced the expression of alpha 4 beta 7, but did not affect the expression of CCR9.
4.LPS- or CpG-DC could enhance the chemotaxis activity of B cells to TECK, which increased by 6.4 and 13.1 percentage points respectively compared with the control group. However, the effect of LPS- or CpG-DC on the chemotaxis activity of B cells to MadCAM-1 was relatively weak, which only increased by 2.5 and 6.25 percentage points respectively.
5.LPS- or CpG-DC promoted B cells to secrete IgA. The IgA concentration in the culture supernatant increased by 4 times and 6 times compared with that in the control group. However, the addition of IL-5 and / or IL-6 did not further promote IgA secretion.
6.LPS or CpG enhanced DC to produce IL-6, TGF- and NO. Beta stimulant concentration in the range of 0.1-10 and cytokine secretion was positively correlated, in a dose-dependent manner.10 g/ml LPS or 10 M CpG after DC2.4 stimulation, the IL-6 secretion were increased by 1.3 and 1.5 times, TGF- beta 1 secretion were increased by 61% and 70%. in 10 g/ml LPS and 10 M CpG, NO secretion reached the highest, compared with the control group, were increased by 47% and 84%.
conclusion
TLR ligands trigger the ability of bone marrow derived DC to produce RA, suggesting that intestinal microbes play a key role in the production of RA in DC.

【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

【共引文献】