探讨p38MAPK信号通路对树突状细胞PD-L1表型的影响

发布时间：2018-07-13 12:06

【摘要】：研究背景冠状动脉粥样硬化性心脏病(冠心病)已经成为威胁人类健康的第三位杀手,也是严重影响人们生活质量的最常见的心血管疾病之一。研究表明冠心病是慢性炎症反应,动脉粥样斑块内有大量的单核细胞、巨噬细胞、树突状细胞和T淋巴细胞等炎症细胞的浸润,斑块内炎症反应非常活跃；而活动性炎症反应可促使稳定性粥样斑块转变为不稳定性斑块,这是冠状动脉内急性血栓形成的诱因。由此可见,抑制炎症细胞活性,阻断炎症反应通路已成为研究冠心病免疫治疗的热点和难点。众所周知,T淋巴细胞的激活是炎症反应的中心环节,而近年来发现的负性刺激信号(PD-1/PD-L1)对调节T淋巴细胞活性起重要作用。程序性死亡因子配体1(programmed cell death ligand1, PD-L1、B7-H1或CD274)是由290个氨基酸构成的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员,它的受体既不是CD28蛋白,也不是CTLA-4蛋白(cytotoxic T-lymphocyte Antigen4, CTLA-4)和ICOS蛋白(inducible co-stimulator, ICOS),其受体为程序性死亡因子1(programmed cell death1receptor, PD-1或CD279)。由于最初在肿瘤细胞中发现PD-L1有高表达,人们因此推测PD-L1可能与肿瘤细胞浸润有关；但随着研究深入,发现除肿瘤细胞以外,PD-Ll在多种炎症细胞和组织细胞上均有表达,如T淋巴细胞、B淋巴细胞、巨噬细胞、Kupffer细胞、星形细胞、树突状细胞、血管内皮细胞、骨髓源性肥大细胞、胎盘合体滋养层细胞等等；PD-L1的功能除与肿瘤细胞浸润以外,还与多种疾病发生有密切的关系,如移植免疫反应、自身免疫性疾病、微生物感染(病毒感染)。近年来研究还发现PD-L1/PD-1与动脉粥样斑块形成有关。Gotsman等研究冠状动脉斑块时,发现用荧光免疫技术可以探测到PD-L1可表达于多处动脉粥样斑块内。Lee等发现冠心病患者外周血T淋巴细胞PD-1表型及树突状细胞PD-L1表型均较健康人的明显降低,而冠心病患者树突状细胞激活初始T淋巴细胞能力明显增强。虽然现在对PD-L1的功能有了一定了解,然而到现在促使细胞表达PD-L1蛋白的分子机制还没有完全清晰。不同研究对象及使用不同刺激物,得出结果不完全相同,归纳起来影响PD-L1表达的细胞信号通路可能为JAK/STAT信号通路、PI3K/Akt信号通路、MEK/Erk信号通路.NPM/ALK通路,以及与IRF-1和STAT-3转录因子有关。众所周知,p38MAPK信号通路是MAPK通路的重要分支,它通过使转录因子磷酸化而改变基因的表达,参与多种细胞内信息传递过程,能对广泛的细胞外信号发生反应,介导细胞生长、发育、分化及死亡全过程。然而关于PD-L1蛋白表达与p38MAPK信号通路的关系的研究资料甚少。鉴于以上证据,本课题以体外培养单核细胞源树突状细胞作为研究对象,利用炎症因子(LPS)刺激树突状细胞模拟病原微生物入侵的模型,观察树突状细胞PD-L1表型的变化；再以p38蛋白特异性抑制剂SB203580阻断p38MAPK通路,探讨树突状细胞表达PD-L1与p38MAPK信号通路的关系,阐明LPS诱导树突状细胞表达PD-L1蛋白的分子机制,完善负性协同刺激信号(PD-1/PD-L)调控T淋巴细胞活性的机理,为动脉粥样硬化的免疫治疗的提供理论基础。第一部分脂多糖诱导单核细胞源树突状细胞表达PD-L1 目的观察炎症因子(LPS)对树突状细胞CD80、CD86和PD-L1表型的影响,以明确LPS能够促进树突状细胞成熟,增强PD-Ll表达的作用。对象和方法 1、对象体外培养健康人外周血单核细胞来源的树突状细胞 2、方法 2、1单核细胞的分离和培养采用密度离心法分离健康人外周血单个核细胞,加入含10%胎牛血清、100U/ml青霉素和100μ g/ml链霉素的RPMI-1640培养基,调整细胞密度为5×106/ml,接种于六孔细胞培养板中,置入饱和湿度、5%CO237℃的细胞孵育箱中培养2小时。取出六孔细胞培养板,吸弃悬浮细胞,即得贴壁的单核细胞。 2、2树突状细胞的诱导和培养贴壁的单核细胞用无Ca2+、Mg2+的PBS轻柔洗2次后,在每个孔加入含rhGM-CSF25ng/ml、rhIL-425ng/ml、100U/ml青霉素和100μ g/ml链霉素的树突状细胞完全培养基约3ml,置于5%CO237℃的孵育箱中继续培养,分别于第3、5天半量换液,补充细胞因子,维持rhGM-CSF和rhIL-4均为25ng/ml。于第7天收获树突状细胞。 2、3实验分组(每组设2个复孔,共实验4次)LPS用二甲基亚砜(DMSO)溶解。根据使用是否使用脂多糖,将实验分为两组：LPS组(LPS)和对照组(NORMAL)。LPS组：树突状细胞给予LPS(1.0μ g/ml)处理后继续培养24小时；对照组：树突状细胞给予DMSO(0.1%V/V)处理后继续培养24小时。 2、4流式细胞术检测细胞表型收集各组树突状细胞,调整细胞浓度为5×105/ml,分别加入相关表型抗体,孵育,清洗2次,用流式细胞仪检测相关表型荧光强度,用Cellquest分析检测结果。 3、统计学方法：所有数据采用SPSS13.0软件处理,计量数据以均数±标准差(x±S)表示,统计学分析采用两独立样本t检验,P0.05认为差异有统计学意义。结果 1、树突状细胞形态学观察倒置显微镜下观察,外周血单核细胞经rhGM-CSF和rhIL-4联合诱导7天,细胞聚集呈克隆分布,体积较大,呈圆形或形状不规则,并有毛刺状突起,为典型树突状细胞形态。LPS组细胞经LPS刺激6小时后贴壁牢固,伪足变为细长,细胞呈梭形；刺激至24小时细胞逐渐恢复圆形,伪足逐渐变短。对照组树突状细胞呈圆形,悬浮生长,伪足较多。 2、流式结果： LPS组树突状细胞CD80表型(1492.46±82.65)、CD86表型(1136.73±81.62)和PD-Ll表型(3665.89±261.66)较对照组的(536.52±64.10,518.47±48.91,1093.38±115.54)均明显增高,组间差异有统计学意义(P值均小于0.01)。结论 1、脂多糖能够促使树突状细胞CD80和CD86表达增高,促进树突状细胞成熟。 2、脂多糖能够诱导树突状细胞PD-Ll表达增高。第二部分p38MAPK通路调控单核细胞源树突状细胞表达PD-L1 目的以p38蛋白特异性抑制剂SB203580阻断p38MAPK信号通路后,观察树突状细胞受炎症因子刺激后PD-Ll表型变化,探讨树突状细胞表达PD-L1与p38MAPK信号通路的关系。对象和方法 1、对象体外培养健康人外周血单核细胞来源的树突状细胞。 2、方法 2、1单核细胞的分离和培养：同第一部分。 2、2树突状细胞的诱导和培养：同第一部分。 2、3实验分组(每组设2个复孔,重复实验4次) 收获未成熟的树突状细胞,调整细胞密度为2×106/ml,接种于6孔培养板中。根据是否使用LPS和p38蛋白特异性抑制剂SB203580将实验分成三组：LPS及SB203580均用二甲基亚砜(DMSO)溶解第一组为LPS刺激组(LPS)：首先在实验细胞中加入DMSO(0.1%V/V)作用1小时,再加入LPS(1.0μ g/ml)继续培养24小时；第二组为SB203580和LPS共刺激组(SB)：首先在实验细胞中加入SB203580(25μ M)作用1小时,再加入LPS(1.0μ g/ml)继续培养24小时；第三组为正常组(NORMAL)将未加入任何药物的细胞继续培养24小时作为阴性对照。2、4流式细胞术检测细胞表型：同第一部分。2、5Western blot检测PD-L1蛋白收获各组细胞,提取蛋白,调节上样量为30μ g总蛋白/个样品,上样,电泳,转膜,加入一抗、二抗,洗膜,染色,曝光,用Gelpro32分析胶片中蛋白条带。 3.统计学方法所有数据采用SPSS13.0软件处理,计量数据以均数±标准差(x±S)表示,统计学分析多样本比较采用单因素方差分析(one-way ANOVA),多重比较采用SNK-q检验,P0.05认为差异有统计学意义。结果 1、树突状细胞形态学改变倒置显微镜下观察,1)LPS组树突状细胞经LPS刺激6小时后贴壁牢固,伪足变为细长,细胞呈梭形；刺激24小时后细胞逐渐恢复圆形,伪足逐渐变短。2)SB组树突状细胞分散,伪足变短或者退化。3)正常组树突状细胞仍旧呈圆形,悬浮生长,伪足较短。 2、细胞表型变化 1)比较三组细胞CDllc平均荧光强度总体均数差异无统计学意义(LPS组：628.19±34.99,SB组：617.44±41.00,正常组：589.68±47.84,F=1.825,P=0.186)。 2)比较三组树突状细胞CD86表型平均荧光强度,三组总体均数差异有统计学意义(F=16.958,P0.01),用SNK-q检验进行两两比较,SB组CD86表型与LPS组的相比明显降低(729.49±48.89vs873.01±71.24,P0.05),与正常组的相比差异无统计学意义(vs736.96±42.11,P0.05),LPS组CD86表型平均荧光强度显著高于正常组的(P0.05)。 3)比较三组PD-L1表型平均荧光强度总体方差不齐,经log10转换后符合方差齐性检验(F=0.152,P=0.86)。分析三组总体均数差异有统计学意义(F=-82.162,P0.01)。两两比较分析,SB组PD-L1表型平均荧光强度(3.03±0.08)明显低于其它两组,与LPS组的相比,P0.01(vs3.51±0.08)；与正常组的相比,P0.05(vs3.18±0.07)。LPS组PD-Ll表型平均荧光强度明显高于正常组的(P0.05)。 3、Western blot检测PD-L1蛋白三组总体方差符合方差齐性检验(F=1.427,P=0.262),三组间均数总体差异有统计学意义(F=75.226,P0.01)；比较组间差异,SB组树突状细胞的PD-L1蛋白含量(0.55±0.08)明显低于其余两组的,与LPS组的相比,P0.05(vs1.24±0.11)；与正常组的相比差异有统计学意义(vs0.95±0.14,P0.05)；LPS组细胞PD-L1蛋白含量高于与正常组(P0.05) 结论 1、抑制p38蛋白后阻断脂多糖的促进树突状细胞成熟作用,说明p38MAPK通路调控树突状细胞成熟。 2、抑制p38蛋白后阻断PD-L1表达增高,p38MAPK通路调控树突状细胞PD-L1表达。
[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.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392

【参考文献】