本文關(guān)鍵詞： 樹突狀細(xì)胞 維甲酸 Toll樣受體 IgA 腸黏膜歸巢　出處：《浙江大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

【摘要】：背景和目的 粘膜是機(jī)體抵抗病原微生物感染的第一道防線,目前發(fā)現(xiàn)90%以上的人類傳染病通過粘膜途徑感染。粘膜免疫在機(jī)體免疫系統(tǒng)中占有重要的地位,它能夠抵抗致病菌的入侵,對飲食和呼吸中的有害抗原,作出適當(dāng)?shù)拿庖邞?yīng)答,同時(shí)對環(huán)境和食物中的無害抗原及共生菌,表現(xiàn)為免疫耐受。所以,如果能夠控制粘膜感染,就可控制大部分傳染病的發(fā)生。 人體腸粘膜總面積為300m2,是機(jī)體內(nèi)外環(huán)境交流的主要界面,是粘膜感染過程中的易發(fā)部位。腸道粘膜免疫系統(tǒng)由腸相關(guān)淋巴組織(gut-associated lymphoid tissue, GALT)組成,是機(jī)體免疫系統(tǒng)中最大最復(fù)雜的網(wǎng)絡(luò)。具有嚴(yán)格的免疫反應(yīng)機(jī)制和完善的免疫調(diào)控機(jī)制。派氏結(jié)(PP)、腸系膜淋巴結(jié)(MLN)及孤立淋巴濾泡是腸道黏膜免疫應(yīng)答的誘導(dǎo)部位,負(fù)責(zé)起始腸道內(nèi)的免疫應(yīng)答；腸道黏膜上皮內(nèi)淋巴細(xì)胞(IEL)及固有層(LP)內(nèi)散在的淋巴細(xì)胞是腸道黏膜免疫應(yīng)答的效應(yīng)細(xì)胞。與系統(tǒng)免疫應(yīng)答相比,粘膜免疫系統(tǒng)的一個(gè)顯著的特點(diǎn)是產(chǎn)生分泌型IgA(secretory IgA, sIgA)。人類腸道每天能夠分泌3g以上的sIgA。已知IgA占體內(nèi)總免疫球蛋白75%以上,其中大部分是位于粘膜表面的sIgA。sIgA對保持腸道上皮的穩(wěn)態(tài)起重要作用,它可與入侵的病原微生物結(jié)合,阻止其在粘膜上皮細(xì)胞表面的定植,并能中和致病菌產(chǎn)生的毒素和侵襲性酶類。此外,sIgA對由食物攝入和空氣吸入的某些抗原物質(zhì)具有免疫排斥作用,可封閉這些抗原,使其游離于粘膜表面不致進(jìn)入機(jī)體,從而避免引起全身的免疫反應(yīng),減少局部過敏反應(yīng)的發(fā)生。 樹突狀細(xì)胞(DC)是功能最強(qiáng)的抗原提呈細(xì)胞,是固有免疫和適應(yīng)性免疫的橋梁和紐帶,其調(diào)節(jié)機(jī)體免疫應(yīng)答的類型和強(qiáng)度。業(yè)已發(fā)現(xiàn)腸道DC有與脾臟、胸腺、腋窩淋巴結(jié)和腹股溝淋巴結(jié)來源的DC不同的特性,前者能產(chǎn)生RA,而后者不產(chǎn)生RA。腸黏膜DC產(chǎn)生的RA是粘膜免疫應(yīng)答的一個(gè)關(guān)鍵性調(diào)節(jié)因素,RA能誘導(dǎo)T、B淋巴細(xì)胞表達(dá)腸黏膜歸巢受體,從而賦予在腸相關(guān)淋巴組織中活化的T、B淋巴細(xì)胞經(jīng)過淋巴細(xì)胞再循環(huán)后,重新歸巢到腸黏膜的特性；RA參與B細(xì)胞IgA類別轉(zhuǎn)換；RA還能影響腸黏膜Treg和Th17的分化,從而可能在炎癥性腸病的發(fā)生和發(fā)展中發(fā)揮作用。但是腸黏膜DC產(chǎn)生RA的機(jī)制——即什么信號(hào)啟動(dòng)DC產(chǎn)生RA迄今尚未闡明。晚近有學(xué)者發(fā)現(xiàn),皮膚和肺中的DC也能產(chǎn)生RA。值得注意的是：能產(chǎn)生RA的DC的組織來源與不產(chǎn)生RA的DC的組織來源相比,前者都與外界環(huán)境相通,存在大量的共生菌,而后者都處于無菌狀態(tài)。所以我們推測,微生物的某些組成成分可能是DC產(chǎn)生RA的始動(dòng)因素。本實(shí)驗(yàn)研究Toll樣受體4和9(TLR4/9)的配體——LPS及CpG對骨髓來源DC合成RA能力的影響。 方法 1.DC細(xì)胞分組處理：培養(yǎng)的骨髓細(xì)胞系——DC2.4經(jīng)PBS洗滌后重懸,以1×106cells/well接種于24孔板,經(jīng)下列三種不同處理,在37℃、5%CO2條件下培養(yǎng)48h。 1.1 LPS對DC產(chǎn)生RA影響的分組：①空白對照組,即DC組；②DC+LPS(10μg/ml)；③DC+LPS(1μg/ml);④DC+LPS(0.1μg/ml);⑤DC+LPS (0.01μg/ml);⑥D(zhuǎn)C+LPS(0.001μg/ml)。 1.2 CpG對DC產(chǎn)生RA影響的分組：①空白對照組,即DC組；②DC+CpG(10μM);③DC+CpG(1μM);④DC+CpG(0.1μM);⑤DC+CpG(0.01μM);⑥D(zhuǎn)C+ CpG(0.001μM)。 1.3 NF-KB阻斷劑SN50對DC產(chǎn)生RA影響的分組：①空白對照組,即DC組;②DC+LPS(1μg/ml);③DC+LPS(1μg/ml)+SN50(18μM);④DC+CpG(1μM);⑤DC+CpG(1μM)+SN50(18μM)。 2.實(shí)時(shí)熒光定量PCR測定處理后DC的RA代謝相關(guān)酶(ALDH1a-1,-2,-3及ADH-1,-4,-5)的表達(dá)。 3.LPS或CpG處理細(xì)胞30和60分鐘后,提取細(xì)胞漿和細(xì)胞核蛋白,Western blotting檢測NF-KB蛋白表達(dá)。在之前培養(yǎng)體系中加入NF-κB蛋白阻斷劑SN50,Western blotting檢測阻斷效應(yīng)。 4.DC和B細(xì)胞共培養(yǎng)：采用流式細(xì)胞術(shù)分選C57BL/6小鼠脾臟B細(xì)胞(5×105),與1μg/ml的LPS或1μM的CpG預(yù)處理48h的DC(1×105)共培養(yǎng)4天。以10種方式處理細(xì)胞(IL-5和IL-6濃度10ng/ml)：空白對照組,即B細(xì)胞組；B+DC2.4細(xì)胞組；B+LPS-DC組;B+LPS-DC+IL-5組;B+LPS-DC+IL-6組;B+LPS-DC+IL-5+IL-6組；B+CpG-DC組；B+CpG-DC+IL-5組;B+CpG-DC+IL-6組;B+CpG-DC+IL-5+IL-6組。 5.收集共培養(yǎng)4天的細(xì)胞,流式細(xì)胞儀分析B細(xì)胞歸巢受體α4β7和CCR9表達(dá)。 6.B細(xì)胞趨化試驗(yàn)：收集與DC共培養(yǎng)后的B細(xì)胞,Transwell細(xì)胞遷移試驗(yàn)檢測B細(xì)胞對趨化因子MadCAM-1和TECK的趨化活性。 7.收集共培養(yǎng)4天的細(xì)胞培養(yǎng)上清,ELISA法檢測上清中IgA濃度。 8. ELISA檢測不同濃度LPS或CpG處理48h的DC培養(yǎng)上清中IL-6和TGF-β1的濃度。 9. Griess法檢測不同濃度LPS或CpG處理48h的DC培養(yǎng)上清中NO濃度。 結(jié)果 1.LPS或CpG能誘導(dǎo)骨髓來源的DC表達(dá)Aldh1a2 mRNA。1μg/ml LPS處理DC48小時(shí)后,Aldh1a2 mRNA表達(dá)增高最為明顯,增高近10倍,ADH4表達(dá)上調(diào)增高18倍。1μM CpG處理DC后Aldh1a2 mRNA表達(dá)提高12.45倍,Aldh1a1 mRNA表達(dá)有近10倍的提高,ADH4表達(dá)上調(diào)近12倍。但無論任一濃度的LPS或CpG處理后,DC Aldh1a3 mRNA都沒有發(fā)現(xiàn)明顯的改變。 2.NF-κB信號(hào)通路在LPS或CpG誘導(dǎo)DC產(chǎn)生RA中發(fā)揮重要作用,用抑制劑SN50阻斷后,NF-κB核轉(zhuǎn)位被抑制,DC表達(dá)Aldh1a2 mRNA下降。SN50加入到LPS-DC (CpG-DC)體系30min后,NF-κB核轉(zhuǎn)位即可被抑制。細(xì)胞核內(nèi)NF-κB/Histone H3比由0.34(0.33)僅上升為0.46(0.48)；60min后阻斷效應(yīng)更加顯著,核內(nèi)NF-κB/Histone H3比0.44(0.47)。SN50使TLR-DC Aldhla2相對表達(dá)量增高幅度分別由10倍之多降為僅2倍。 3.LPS-或CpG-DC增強(qiáng)B細(xì)胞腸黏膜歸巢受體α4β7和CCR9的表達(dá),其中上調(diào)CCR9較α4β7明顯,CCR9平均熒光強(qiáng)度均提高了1倍之多。IL-5和/或IL-6增強(qiáng)α4β7表達(dá),但不影響CCR9表達(dá)。 4.LPS-或CpG-DC能增強(qiáng)B細(xì)胞對TECK的趨化活性,與對照組相比,分別提高了6.4和13.1個(gè)百分點(diǎn)。但LPS-或CpG-DC對B細(xì)胞對MadCAM-1的趨化活性影響較弱,僅分別提高2.5和6.25個(gè)百分點(diǎn)。 5.LPS-或CpG-DC促進(jìn)B細(xì)胞分泌IgA,培養(yǎng)上清中IgA濃度與對照組相比分別提高近4倍和6倍。但外加的IL-5和/或IL-6并沒有進(jìn)一步促進(jìn)IgA的分泌。 6.LPS或CpG增強(qiáng)DC產(chǎn)生IL-6,TGF-β和NO的能力。刺激物濃度在0.1-10區(qū)間內(nèi)與細(xì)胞因子分泌成正相關(guān),呈現(xiàn)劑量依賴性。10μg/ml LPS或10μM CpG刺激DC2.4后,其IL-6分泌量分別提高了1.3和1.5倍,TGF-β1分泌量分別增加了61%和70%。在10μg/ml LPS及10μM CpG時(shí),NO分泌量達(dá)到最高,與對照組相比,分別提高了47%和84%。 結(jié)論 TLR配體能觸發(fā)骨髓來源DC獲得產(chǎn)生RA的能力,提示腸道微生物在DC產(chǎn)生RA中發(fā)揮關(guān)鍵作用。
[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.

【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R392

【共引文獻(xiàn)】

相關(guān)博士學(xué)位論文 前1條

1 隋春陽;TLR4受體信號(hào)傳導(dǎo)通路參與人肝癌細(xì)胞株HepG-2分泌細(xì)胞因子機(jī)制的研究[D];中國醫(yī)科大學(xué);2007年

相關(guān)碩士學(xué)位論文 前1條

1 孫安娜;腫瘤微環(huán)境誘導(dǎo)不成熟樹突狀細(xì)胞為調(diào)節(jié)性樹突狀細(xì)胞的研究[D];浙江大學(xué);2007年

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本文編號(hào)：1448242