【摘要】：研究背景 冠狀動(dòng)脈粥樣硬化性心臟病(冠心病)已經(jīng)成為威脅人類健康的第三位殺手,也是嚴(yán)重影響人們生活質(zhì)量的最常見的心血管疾病之一。研究表明冠心病是慢性炎癥反應(yīng),動(dòng)脈粥樣斑塊內(nèi)有大量的單核細(xì)胞、巨噬細(xì)胞、樹突狀細(xì)胞和T淋巴細(xì)胞等炎癥細(xì)胞的浸潤(rùn),斑塊內(nèi)炎癥反應(yīng)非�；钴S；而活動(dòng)性炎癥反應(yīng)可促使穩(wěn)定性粥樣斑塊轉(zhuǎn)變?yōu)椴环�(wěn)定性斑塊,這是冠狀動(dòng)脈內(nèi)急性血栓形成的誘因。由此可見,抑制炎癥細(xì)胞活性,阻斷炎癥反應(yīng)通路已成為研究冠心病免疫治療的熱點(diǎn)和難點(diǎn)。眾所周知,T淋巴細(xì)胞的激活是炎癥反應(yīng)的中心環(huán)節(jié),而近年來發(fā)現(xiàn)的負(fù)性刺激信號(hào)(PD-1/PD-L1)對(duì)調(diào)節(jié)T淋巴細(xì)胞活性起重要作用。 程序性死亡因子配體1(programmed cell death ligand1, PD-L1、B7-H1或CD274)是由290個(gè)氨基酸構(gòu)成的Ⅰ型跨膜蛋白。早在1999年Dong等研究指出PD-L1是B7家族分子的第三位成員,它的受體既不是CD28蛋白,也不是CTLA-4蛋白(cytotoxic T-lymphocyte Antigen4, CTLA-4)和ICOS蛋白(inducible co-stimulator, ICOS),其受體為程序性死亡因子1(programmed cell death1receptor, PD-1或CD279)。由于最初在腫瘤細(xì)胞中發(fā)現(xiàn)PD-L1有高表達(dá),人們因此推測(cè)PD-L1可能與腫瘤細(xì)胞浸潤(rùn)有關(guān)；但隨著研究深入,發(fā)現(xiàn)除腫瘤細(xì)胞以外,PD-Ll在多種炎癥細(xì)胞和組織細(xì)胞上均有表達(dá),如T淋巴細(xì)胞、B淋巴細(xì)胞、巨噬細(xì)胞、Kupffer細(xì)胞、星形細(xì)胞、樹突狀細(xì)胞、血管內(nèi)皮細(xì)胞、骨髓源性肥大細(xì)胞、胎盤合體滋養(yǎng)層細(xì)胞等等；PD-L1的功能除與腫瘤細(xì)胞浸潤(rùn)以外,還與多種疾病發(fā)生有密切的關(guān)系,如移植免疫反應(yīng)、自身免疫性疾病、微生物感染(病毒感染)。近年來研究還發(fā)現(xiàn)PD-L1/PD-1與動(dòng)脈粥樣斑塊形成有關(guān)。Gotsman等研究冠狀動(dòng)脈斑塊時(shí),發(fā)現(xiàn)用熒光免疫技術(shù)可以探測(cè)到PD-L1可表達(dá)于多處動(dòng)脈粥樣斑塊內(nèi)。Lee等發(fā)現(xiàn)冠心病患者外周血T淋巴細(xì)胞PD-1表型及樹突狀細(xì)胞PD-L1表型均較健康人的明顯降低,而冠心病患者樹突狀細(xì)胞激活初始T淋巴細(xì)胞能力明顯增強(qiáng)。雖然現(xiàn)在對(duì)PD-L1的功能有了一定了解,然而到現(xiàn)在促使細(xì)胞表達(dá)PD-L1蛋白的分子機(jī)制還沒有完全清晰。不同研究對(duì)象及使用不同刺激物,得出結(jié)果不完全相同,歸納起來影響PD-L1表達(dá)的細(xì)胞信號(hào)通路可能為JAK/STAT信號(hào)通路、PI3K/Akt信號(hào)通路、MEK/Erk信號(hào)通路.NPM/ALK通路,以及與IRF-1和STAT-3轉(zhuǎn)錄因子有關(guān)。眾所周知,p38MAPK信號(hào)通路是MAPK通路的重要分支,它通過使轉(zhuǎn)錄因子磷酸化而改變基因的表達(dá),參與多種細(xì)胞內(nèi)信息傳遞過程,能對(duì)廣泛的細(xì)胞外信號(hào)發(fā)生反應(yīng),介導(dǎo)細(xì)胞生長(zhǎng)、發(fā)育、分化及死亡全過程。然而關(guān)于PD-L1蛋白表達(dá)與p38MAPK信號(hào)通路的關(guān)系的研究資料甚少。 鑒于以上證據(jù),本課題以體外培養(yǎng)單核細(xì)胞源樹突狀細(xì)胞作為研究對(duì)象,利用炎癥因子(LPS)刺激樹突狀細(xì)胞模擬病原微生物入侵的模型,觀察樹突狀細(xì)胞PD-L1表型的變化；再以p38蛋白特異性抑制劑SB203580阻斷p38MAPK通路,探討樹突狀細(xì)胞表達(dá)PD-L1與p38MAPK信號(hào)通路的關(guān)系,闡明LPS誘導(dǎo)樹突狀細(xì)胞表達(dá)PD-L1蛋白的分子機(jī)制,完善負(fù)性協(xié)同刺激信號(hào)(PD-1/PD-L)調(diào)控T淋巴細(xì)胞活性的機(jī)理,為動(dòng)脈粥樣硬化的免疫治療的提供理論基礎(chǔ)。 第一部分脂多糖誘導(dǎo)單核細(xì)胞源樹突狀細(xì)胞表達(dá)PD-L1 目的觀察炎癥因子(LPS)對(duì)樹突狀細(xì)胞CD80、CD86和PD-L1表型的影響,以明確LPS能夠促進(jìn)樹突狀細(xì)胞成熟,增強(qiáng)PD-Ll表達(dá)的作用。 對(duì)象和方法 1、對(duì)象體外培養(yǎng)健康人外周血單核細(xì)胞來源的樹突狀細(xì)胞 2、方法 2、1單核細(xì)胞的分離和培養(yǎng) 采用密度離心法分離健康人外周血單個(gè)核細(xì)胞,加入含10%胎牛血清、100U/ml青霉素和100μ g/ml鏈霉素的RPMI-1640培養(yǎng)基,調(diào)整細(xì)胞密度為5×106/ml,接種于六孔細(xì)胞培養(yǎng)板中,置入飽和濕度、5%CO237℃的細(xì)胞孵育箱中培養(yǎng)2小時(shí)。取出六孔細(xì)胞培養(yǎng)板,吸棄懸浮細(xì)胞,即得貼壁的單核細(xì)胞。 2、2樹突狀細(xì)胞的誘導(dǎo)和培養(yǎng) 貼壁的單核細(xì)胞用無Ca2+、Mg2+的PBS輕柔洗2次后,在每個(gè)孔加入含rhGM-CSF25ng/ml、rhIL-425ng/ml、100U/ml青霉素和100μ g/ml鏈霉素的樹突狀細(xì)胞完全培養(yǎng)基約3ml,置于5%CO237℃的孵育箱中繼續(xù)培養(yǎng),分別于第3、5天半量換液,補(bǔ)充細(xì)胞因子,維持rhGM-CSF和rhIL-4均為25ng/ml。于第7天收獲樹突狀細(xì)胞。 2、3實(shí)驗(yàn)分組(每組設(shè)2個(gè)復(fù)孔,共實(shí)驗(yàn)4次)LPS用二甲基亞砜(DMSO)溶解。 根據(jù)使用是否使用脂多糖,將實(shí)驗(yàn)分為兩組：LPS組(LPS)和對(duì)照組(NORMAL)。LPS組：樹突狀細(xì)胞給予LPS(1.0μ g/ml)處理后繼續(xù)培養(yǎng)24小時(shí)；對(duì)照組：樹突狀細(xì)胞給予DMSO(0.1%V/V)處理后繼續(xù)培養(yǎng)24小時(shí)。 2、4流式細(xì)胞術(shù)檢測(cè)細(xì)胞表型 收集各組樹突狀細(xì)胞,調(diào)整細(xì)胞濃度為5×105/ml,分別加入相關(guān)表型抗體,孵育,清洗2次,用流式細(xì)胞儀檢測(cè)相關(guān)表型熒光強(qiáng)度,用Cellquest分析檢測(cè)結(jié)果。 3、統(tǒng)計(jì)學(xué)方法： 所有數(shù)據(jù)采用SPSS13.0軟件處理,計(jì)量數(shù)據(jù)以均數(shù)±標(biāo)準(zhǔn)差(x±S)表示,統(tǒng)計(jì)學(xué)分析采用兩獨(dú)立樣本t檢驗(yàn),P0.05認(rèn)為差異有統(tǒng)計(jì)學(xué)意義。 結(jié)果 1、樹突狀細(xì)胞形態(tài)學(xué)觀察 倒置顯微鏡下觀察,外周血單核細(xì)胞經(jīng)rhGM-CSF和rhIL-4聯(lián)合誘導(dǎo)7天,細(xì)胞聚集呈克隆分布,體積較大,呈圓形或形狀不規(guī)則,并有毛刺狀突起,為典型樹突狀細(xì)胞形態(tài)。LPS組細(xì)胞經(jīng)LPS刺激6小時(shí)后貼壁牢固,偽足變?yōu)榧?xì)長(zhǎng),細(xì)胞呈梭形；刺激至24小時(shí)細(xì)胞逐漸恢復(fù)圓形,偽足逐漸變短。對(duì)照組樹突狀細(xì)胞呈圓形,懸浮生長(zhǎng),偽足較多。 2、流式結(jié)果： LPS組樹突狀細(xì)胞CD80表型(1492.46±82.65)、CD86表型(1136.73±81.62)和PD-Ll表型(3665.89±261.66)較對(duì)照組的(536.52±64.10,518.47±48.91,1093.38±115.54)均明顯增高,組間差異有統(tǒng)計(jì)學(xué)意義(P值均小于0.01)。 結(jié)論 1、脂多糖能夠促使樹突狀細(xì)胞CD80和CD86表達(dá)增高,促進(jìn)樹突狀細(xì)胞成熟。 2、脂多糖能夠誘導(dǎo)樹突狀細(xì)胞PD-Ll表達(dá)增高。 第二部分p38MAPK通路調(diào)控單核細(xì)胞源樹突狀細(xì)胞表達(dá)PD-L1 目的以p38蛋白特異性抑制劑SB203580阻斷p38MAPK信號(hào)通路后,觀察樹突狀細(xì)胞受炎癥因子刺激后PD-Ll表型變化,探討樹突狀細(xì)胞表達(dá)PD-L1與p38MAPK信號(hào)通路的關(guān)系。 對(duì)象和方法 1、對(duì)象體外培養(yǎng)健康人外周血單核細(xì)胞來源的樹突狀細(xì)胞。 2、方法 2、1單核細(xì)胞的分離和培養(yǎng)：同第一部分。 2、2樹突狀細(xì)胞的誘導(dǎo)和培養(yǎng)：同第一部分。 2、3實(shí)驗(yàn)分組(每組設(shè)2個(gè)復(fù)孔,重復(fù)實(shí)驗(yàn)4次) 收獲未成熟的樹突狀細(xì)胞,調(diào)整細(xì)胞密度為2×106/ml,接種于6孔培養(yǎng)板中。根據(jù)是否使用LPS和p38蛋白特異性抑制劑SB203580將實(shí)驗(yàn)分成三組：LPS及SB203580均用二甲基亞砜(DMSO)溶解 第一組為L(zhǎng)PS刺激組(LPS)：首先在實(shí)驗(yàn)細(xì)胞中加入DMSO(0.1%V/V)作用1小時(shí),再加入LPS(1.0μ g/ml)繼續(xù)培養(yǎng)24小時(shí)； 第二組為SB203580和LPS共刺激組(SB)：首先在實(shí)驗(yàn)細(xì)胞中加入SB203580(25μ M)作用1小時(shí),再加入LPS(1.0μ g/ml)繼續(xù)培養(yǎng)24小時(shí)； 第三組為正常組(NORMAL)將未加入任何藥物的細(xì)胞繼續(xù)培養(yǎng)24小時(shí)作為陰性對(duì)照。2、4流式細(xì)胞術(shù)檢測(cè)細(xì)胞表型：同第一部分。2、5Western blot檢測(cè)PD-L1蛋白 收獲各組細(xì)胞,提取蛋白,調(diào)節(jié)上樣量為30μ g總蛋白/個(gè)樣品,上樣,電泳,轉(zhuǎn)膜,加入一抗、二抗,洗膜,染色,曝光,用Gelpro32分析膠片中蛋白條帶。 3.統(tǒng)計(jì)學(xué)方法 所有數(shù)據(jù)采用SPSS13.0軟件處理,計(jì)量數(shù)據(jù)以均數(shù)±標(biāo)準(zhǔn)差(x±S)表示,統(tǒng)計(jì)學(xué)分析多樣本比較采用單因素方差分析(one-way ANOVA),多重比較采用SNK-q檢驗(yàn),P0.05認(rèn)為差異有統(tǒng)計(jì)學(xué)意義。 結(jié)果 1、樹突狀細(xì)胞形態(tài)學(xué)改變 倒置顯微鏡下觀察,1)LPS組樹突狀細(xì)胞經(jīng)LPS刺激6小時(shí)后貼壁牢固,偽足變?yōu)榧?xì)長(zhǎng),細(xì)胞呈梭形；刺激24小時(shí)后細(xì)胞逐漸恢復(fù)圓形,偽足逐漸變短。2)SB組樹突狀細(xì)胞分散,偽足變短或者退化。3)正常組樹突狀細(xì)胞仍舊呈圓形,懸浮生長(zhǎng),偽足較短。 2、細(xì)胞表型變化 1)比較三組細(xì)胞CDllc平均熒光強(qiáng)度總體均數(shù)差異無統(tǒng)計(jì)學(xué)意義(LPS組：628.19±34.99,SB組：617.44±41.00,正常組：589.68±47.84,F=1.825,P=0.186)。 2)比較三組樹突狀細(xì)胞CD86表型平均熒光強(qiáng)度,三組總體均數(shù)差異有統(tǒng)計(jì)學(xué)意義(F=16.958,P0.01),用SNK-q檢驗(yàn)進(jìn)行兩兩比較,SB組CD86表型與LPS組的相比明顯降低(729.49±48.89vs873.01±71.24,P0.05),與正常組的相比差異無統(tǒng)計(jì)學(xué)意義(vs736.96±42.11,P0.05),LPS組CD86表型平均熒光強(qiáng)度顯著高于正常組的(P0.05)。 3)比較三組PD-L1表型平均熒光強(qiáng)度總體方差不齊,經(jīng)log10轉(zhuǎn)換后符合方差齊性檢驗(yàn)(F=0.152,P=0.86)。分析三組總體均數(shù)差異有統(tǒng)計(jì)學(xué)意義(F=-82.162,P0.01)。兩兩比較分析,SB組PD-L1表型平均熒光強(qiáng)度(3.03±0.08)明顯低于其它兩組,與LPS組的相比,P0.01(vs3.51±0.08)；與正常組的相比,P0.05(vs3.18±0.07)。LPS組PD-Ll表型平均熒光強(qiáng)度明顯高于正常組的(P0.05)。 3、Western blot檢測(cè)PD-L1蛋白 三組總體方差符合方差齊性檢驗(yàn)(F=1.427,P=0.262),三組間均數(shù)總體差異有統(tǒng)計(jì)學(xué)意義(F=75.226,P0.01)；比較組間差異,SB組樹突狀細(xì)胞的PD-L1蛋白含量(0.55±0.08)明顯低于其余兩組的,與LPS組的相比,P0.05(vs1.24±0.11)；與正常組的相比差異有統(tǒng)計(jì)學(xué)意義(vs0.95±0.14,P0.05)；LPS組細(xì)胞PD-L1蛋白含量高于與正常組(P0.05) 結(jié)論 1、抑制p38蛋白后阻斷脂多糖的促進(jìn)樹突狀細(xì)胞成熟作用,說明p38MAPK通路調(diào)控樹突狀細(xì)胞成熟。 2、抑制p38蛋白后阻斷PD-L1表達(dá)增高,p38MAPK通路調(diào)控樹突狀細(xì)胞PD-L1表達(dá)。
[Abstract]:Research background
Coronary atherosclerotic heart disease (CHD) has become a third killer that threatens human health and is one of the most common cardiovascular diseases that seriously affect people's quality of life. The study shows that coronary heart disease is a chronic inflammatory reaction. There are a large number of monocytes, macrophages, dendritic cells, and T lymphatic cells in atherosclerotic plaque. Inflammatory reaction in the plaque is very active in the cell, and the active inflammatory response can induce the stability of the atherosclerotic plaque to turn into unstable plaque, which is the cause of acute coronary thrombosis in the coronary artery. Thus, it can be seen that inhibiting the activity of inflammatory cells and blocking the inflammatory response pathway have become the heat of the study of coronary heart disease. As we all know, the activation of T lymphocytes is the central link of the inflammatory response, and the negative stimulation signal (PD-1/PD-L1), discovered in recent years, plays an important role in regulating the activity of T lymphocytes.
The programmed death factor ligand 1 (programmed cell death ligand1, PD-L1, B7-H1 or CD274) is a type I transmembrane protein composed of 290 amino acids. Early in 1999 Dong and other studies indicated that PD-L1 is the third member of the B7 family molecule, and its receptor is neither CD28 protein nor CTLA-4 protein. ICOS protein (inducible co-stimulator, ICOS), its receptor is programmed cell death factor 1 (programmed cell death1receptor, PD-1 or CD279). Due to the initial high expression of PD-L1 in tumor cells, it is presumed that PD-L1 may be associated with tumor cell infiltration, but as the study goes deep, PD-Ll is more than tumor cells. The expression of T lymphocytes, B lymphocytes, macrophages, macrophages, Kupffer cells, astrocytes, dendritic cells, vascular endothelial cells, marrow derived mast cells, placental syncytio cells, and so on. The function of PD-L1 is closely related to the occurrence of a variety of diseases, except for the infiltration of tumor cells. In recent years, we found that when PD-L1/PD-1 and atherosclerotic plaque form.Gotsman and other coronary atherosclerotic plaques, it was found that PD-L1 could be detected by.Lee in multiple atherosclerotic plaques and found in patients with coronary artery disease. The PD-1 phenotype of T lymphocytes in peripheral blood and the PD-L1 phenotype of dendritic cells were significantly lower than those of the healthy people, while the ability to activate the initial T lymphocyte in the dendritic cells of the patients with coronary heart disease was obviously enhanced. Although the function of PD-L1 was known to a certain extent, the molecular mechanism of promoting the expression of PD-L1 protein has not yet been completely clear. The results of different subjects and different stimuli are not exactly the same. The cell signaling pathways that induce PD-L1 expression may be JAK/STAT signaling pathway, PI3K/Akt signaling pathway, MEK/Erk signaling pathway.NPM/ALK pathway, and IRF-1 and STAT-3 transcription factors. It is well known that the p38MAPK signaling pathway is the weight of the MAPK pathway. To branching, it changes the expression of genes by phosphorylating the transcription factors and participates in a variety of intracellular information transmission processes. It can react to a wide range of extracellular signals and mediate the whole process of cell growth, development, differentiation and death. However, little research has been made about the relationship between the expression of PD-L1 protein and the p38MAPK signaling pathway.
In view of the above evidence, this subject uses dendritic cells derived from mononuclear cells as the research object in vitro, and uses LPS to stimulate dendritic cells to simulate the model of pathogenic microorganism invasion and observe the changes in PD-L1 phenotype of dendritic cells, and then block the p38MAPK pathway with the specific inhibitor SB203580 of p38 protein to explore the dendritic cells. To express the relationship between PD-L1 and p38MAPK signaling pathway, to clarify the molecular mechanism of LPS induced PD-L1 protein expression in dendritic cells, to improve the mechanism of negative co stimulation signal (PD-1/PD-L) to regulate the activity of T lymphocytes, and to provide a theoretical basis for the immunotherapy of atherosclerosis.
Part 1 lipopolysaccharide induces monocyte derived dendritic cells to express PD-L1
Objective To observe the effect of inflammatory factors (LPS) on the phenotype of CD80, CD86 and PD-L1 in dendritic cells, so that LPS can promote the maturation of dendritic cells and enhance the expression of PD-Ll.
Objects and methods
1, in vitro culture of dendritic cells derived from healthy human peripheral blood mononuclear cells.
2, method
Isolation and culture of 2,1 mononuclear cells
The density centrifugation method was used to separate the peripheral blood mononuclear cells of healthy people, add the RPMI-1640 medium containing 10% fetal bovine serum, 100U/ml penicillin and 100 g/ml streptomycin, adjust the cell density to 5 x 106/ml, inoculate in the six pore cell culture plate, put into the saturated humidity and incubate the cell incubating box for 2 hours at 5%CO237 centigrade, and take out the culture of six pore cells. Boards, which suck up suspended cells, have adherent mononuclear cells.
Induction and culture of 2,2 dendritic cells
After the adherent mononuclear cells were washed gently for 2 times without Ca2+, Mg2+ PBS, the dendritic cells containing rhGM-CSF25ng/ml, rhIL-425ng/ml, 100U/ml penicillin and streptomycin were completely cultured on the basal 3ml, and kept in the incubators at 5%CO237 centigrade, to replace the liquid in the first half of the 3,5 days, supplemented by cytokines, and maintained rhGM-CSF. RhIL-4 was 25ng/ml., and the dendritic cells were harvested on the seventh day.
2,3 experimental group (2 holes in each group, 4 experiments), LPS was dissolved with two methyl sulfoxide (DMSO).
According to the use of lipopolysaccharide, the experiment was divided into two groups: LPS group (LPS) and control group (NORMAL).LPS group: dendritic cells were treated with LPS (1 g/ml) for 24 hours; control group: dendritic cells were treated for 24 hours after DMSO (0.1%V/V) treatment.
Detection of cell phenotype by 2,4 flow cytometry
Each group of dendritic cells was collected, and the cell concentration was 5 x 105/ml. The related phenotypic antibodies were added to the cells respectively. They were incubated and cleaned 2 times. The fluorescence intensity of the related phenotypes was detected by flow cytometry, and the results were analyzed by Cellquest.
3, statistical methods:
All data were treated with SPSS13.0 software, and the measured data were expressed as mean standard deviation (x + S). Statistical analysis used two independent samples t test. P0.05 thought the difference was statistically significant.
Result
1, morphological observation of dendritic cells
Under the inverted microscope, the peripheral blood mononuclear cells were induced by rhGM-CSF and rhIL-4 for 7 days. The cell aggregation was cloned and distributed, the volume was large, the cells were round or irregular in shape, and had spur shaped protuberances. The cell morphology of the typical dendritic cells was strong for 6 hours after the stimulation of the cells in the.LPS group, and the cells were elongated and spindle shaped. The cells were stimulated to 2. At 4 hours, the cells gradually recovered round and the pseudo foot became shorter. The dendritic cells in the control group were round, suspending and growing.
2, flow results:
The CD80 phenotypes of dendritic cells in group LPS (1492.46 + 82.65), CD86 phenotypes (1136.73 + 81.62) and PD-Ll phenotypes (3665.89 + 261.66) were significantly higher than those of the control group (536.52 + 64.10518.47 + 48.911093.38 + 115.54), and there was a significant difference between the groups (P value was less than 0.01).
conclusion
1, lipopolysaccharide can increase the expression of CD80 and CD86 in dendritic cells and promote the maturation of dendritic cells.
2, lipopolysaccharide can induce increased expression of PD-Ll in dendritic cells.
The second part of the p38MAPK pathway regulates the expression of PD-L1 in monocyte derived dendritic cells.
Objective To observe the PD-Ll phenotype of dendritic cells stimulated by inflammatory factors after blocking the p38MAPK signaling pathway of p38 protein specific inhibitor SB203580, and to explore the relationship between the expression of PD-L1 and the p38MAPK signaling pathway in dendritic cells.
Objects and methods
1, cultured dendritic cells derived from peripheral blood mononuclear cells from healthy individuals were cultured in vitro.
2, method
Isolation and culture of 2,1 monocytes: Part one.
Induction and culture of 2,2 dendritic cells: same as the first part.
2,3 experimental group (each group consisted of 2 duplicate holes and 4 repeated experiments).
Immature dendritic cells were harvested with a cell density of 2 x 106/ml and inoculated in 6 Hole culture plates. The experiment was divided into three groups based on whether LPS and p38 protein specific inhibitor SB203580 were used: LPS and SB203580 were dissolved with two methyl sulfoxide (DMSO).
The first group was the LPS stimulation group (LPS). First, DMSO (0.1%V/V) was added to the experimental cells for 1 hours, then LPS (1 g/ml) was added to the experiment for 24 hours.
The second groups were SB203580 and LPS co stimulation group (SB): first, SB203580 (25 mu M) was added to the experimental cells for 1 hours, and then added to LPS (1 mu g/ml) for 24 hours.
In the third group, the cells in the normal group (NORMAL) continued to be cultured for 24 hours without any drug. The cell phenotype was detected by the negative control.2,4 flow cytometry, and the first part.2,5Western blot was used to detect the PD-L1 protein.
The cells were harvested, the protein was extracted, the sample was adjusted to 30 mu g total protein / sample, sample, electrophoresis, and membrane, adding one resistance, two resistance, washing, dyeing, exposure, and Gelpro32 analysis of the protein strips in the film.
3. statistical method
All data were treated with SPSS13.0 software, and the measured data were expressed with mean standard deviation (x + S). The statistical analysis was compared with single factor analysis of variance (one-way ANOVA), and multiple comparison used SNK-q test. P0.05 thought the difference was statistically significant.
Result
1, morphological changes of dendritic cells
Under the inverted microscope, 1) 1) the dendritic cells in the LPS group were firmly adhered to the wall after 6 hours of stimulation, the pseudo foot became slender, the cells were spindle shaped, the cells gradually resumed round and the pseudo foot gradually shortened to.2 after 24 hours of stimulation. The dendritic cells in the SB group were dispersed, the pseudo foot became short or degraded.3.) the dendritic cells in the normal group were still round, suspended and compared with the pseudo foot. Short.
2, cell phenotypic change
1) there was no significant difference in the average fluorescence intensity of CDllc in the three groups (group LPS: 628.19 + 34.99, SB group: 617.44 + 41, 589.68 + 47.84, F=1.825, P=0.186).
2) compared the average fluorescence intensity of CD86 phenotypes in three groups of dendritic cells, the difference between the three groups was statistically significant (F=16.958, P0.01), and the CD86 phenotype in the SB group was significantly lower than that in the LPS group (729.49 + 48.89vs873.01 + 71.24, P0.05), and there was no statistically significant difference compared with the normal group (vs736.96 + 42.11, P0.05). The mean fluorescence intensity of CD86 phenotype in group LPS was significantly higher than that in normal group (P0.05).
3) the overall variance of the average fluorescence intensity of the three groups of PD-L1 phenotypes was not homogeneous. After log10 conversion, it was consistent with the homogeneity test of variance (F=0.152, P=0.86). The difference between the three groups was statistically significant (F=-82.162, P0.01). 22 comparative analysis of the average fluorescence intensity of PD-L1 phenotypes in SB group (3.03 + 0.08) was significantly lower than that of the other two groups, compared with the LPS group, P0.0. 1 (vs3.51 + 0.08); compared with the normal group, the mean fluorescence intensity of PD-Ll phenotype in P0.05 (vs3.18 + 0.07).LPS group was significantly higher than that in normal group (P0.05).
3, Western blot detection of PD-L1 protein
The total variance of the three groups conforms to the homogeneity test of variance (F=1.427, P=0.262). The overall difference between the three groups was statistically significant (F=75.226, P0.01), and the difference between the groups of the SB group (0.55 + 0.08) was significantly lower than the other two groups, and compared with the LPS group, P0.05 (vs1.24 + 0.11); compared with the normal group, there was a difference. The significance of study was (vs0.95 + 0.14, P0.05); the content of PD-L1 protein in group LPS was higher than that in normal group (P0.05).
conclusion
1, blocking p38 protein after blocking lipopolysaccharide promotes dendritic cell maturation, indicating that p38MAPK pathway regulates dendritic cell maturation.
2, inhibition of p38 protein blocks PD-L1 expression and p38MAPK pathway regulates PD-L1 expression in dendritic cells.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R392

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