血管紧张素Ⅱ对人外周单核细胞源树突状细胞免疫功能的影响及机制研究

发布时间：2018-03-28 11:37

本文选题：动脉粥样硬化　切入点：树突状细胞　出处：《浙江大学》2008年博士论文

【摘要】： 【背景和目的】目前认为AS是一种慢性炎症反应过程,增厚的病灶是血管壁内结缔组织对致炎因子的一种增生性反应,是各种特异的细胞、分子对外源性的危险因子与抗原的反应。树突状细胞是一种广泛分布于机体各组织的抗原递呈细胞,近来认为DC可能处于AS免疫反应的中心地位。大量的研究发现肾素-血管紧张素系统可能参与了动脉粥样硬化的发生发展过程。基础研究发现AngⅡ通过AT1受体刺激炎症介质的分泌、诱导细胞凋亡、促进ox-LDL的摄取、产生氧自由基和影响纤溶功能等多方面参与动脉粥样硬化过程。其中刺激炎症介质的分泌是最重要的机制之一,甚至有研究者提出AngⅡ为特殊的细胞因子。以往的研究发现AngⅡ对多种斑块内的细胞成分(内皮细胞、平滑肌细胞、巨噬细胞)均有促炎症介质分泌的作用:刺激内皮细胞表达黏附分子及增加单核细胞对内皮的黏附而促进斑块的发展,诱导内皮细胞表达MMP-2;诱导单核或巨噬细胞产生氧自由基;诱导平滑肌细胞分泌IL-6、MCP-1,促进迁移。而AngⅡ对抗原递呈细胞DC的研究则较少,其具体的作用及机制尚不清楚。有研究曾报道DC表达血管紧张素原、血管紧张素转换酶、血管紧张素Ⅱ受体随其成熟而改变,推测RAS系统可能促进DC表达炎症因子。AngⅡ促进单核细胞向DC的分化,ARB氯沙坦能阻断这种作用而使单核细胞向巨噬样细胞分化。作为RAS系统中居于核心位置的AngⅡ能否作为致AS抗原物诱导DC的免疫成熟,从而增强激活T细胞的能力,促进斑块的形成,目前尚无定论。如果AngⅡ能诱导DC的免疫成熟,促进炎症介质的分泌,其机制又如何?本研究的目的是利用体外培养人单核细胞源性的树突状细胞,评价AngⅡ对DC免疫功能的影响,并对其中的机制进行初探,为防治动脉粥样硬化提供新的思路。【实验方法】 1.细胞的分离和培养:用淋巴细胞分离液从健康成人外周血白细胞悬液分离单个核细胞,再用CD14~+磁珠分选出纯度大于98%的CD14~+单核细胞,用含20ng/ml rhGM-CSF、10ng/ml rhIL-4的RPMI1640培养基培养,第5天收集悬浮细胞作为未成熟DC(immature DC,imDC),加100ng/ml的LPS继续培养24小时收集起来即为成熟DC(mature DC,mDC)。 2.流式细胞学测定DC表面分子的表达。 3.混合淋巴细胞反应测定DC的促淋巴细胞增殖的能力。 4.吞噬实验测定DC的吞噬功能。 5.ELISA测定细胞培养上清中细胞因子。 6.半定量RT-PCR测定树突状细胞趋化因子的表达。 7.采用Western印迹测定磷酸化ERK1/2、p38及TLR4的表达。 8.采用EMSA法测定核内NF-κB活性水平。【结果】 1.AngⅡ组细胞表达CD40较对照组增高,CD86、CD83、HLA-DR的表达则无明显差异,V+AngⅡ组CD40与对照组无差异。LPS刺激下,AngⅡ组与对照组细胞表面分子的表达无明显差异。 2.AngⅡ组DC的吞噬、促异种T淋巴细胞增殖的能力与对照组相比无明显差异。LPS刺激下,AngⅡ组子代T细胞比例高于对照组,V+AngⅡ组与对照组无差异。 3.AngⅡ干预后的DC培养上清中IL-6、TNF-α明显高于对照组,Valsartan能抑制血管紧张素Ⅱ诱导的IL-6、TNF-α分泌。LPS刺激下,AngⅡ组DC培养上清中TNF-α和IL-12均高于对照组,V+AngⅡ组与对照组无差异。 4.AngⅡ干预后的DC表达MCP-1、RANTES、MIP-1α增加,Valsartan能抑制AngⅡ诱导的MCP-1、MIP-1α表达,但不抑制RANTES的表达。LPS刺激下,AngⅡ组DC MCP-1、RANTES、MIP-1α、IP-10的表达均高于对照组,V+AngⅡ组与对照组无差异。 5.AngⅡ刺激磷酸化ERK1/2、p38的表达,ERK1/2、p38的抑制剂能抑制AngⅡ诱导的IL-6、TNF-α的分泌以及MCP-1、RANTES、MIP-1α的表达。 6.AngⅡ可显著增加DC NF-κB的活化,Valsartan能抑制AngⅡ诱导的NF-κB活化。 7.LPS刺激下,AngⅡ组5min、15min的磷酸化ERK1/2的水平高于对照组,Valsartan能抑制AngⅡ增强的磷酸化ERK1/2表达。AngⅡ组与对照组磷酸化p38的水平无明显差异。 8.浓度为0.0001-1μmol/L的AngⅡ干预24h,TLR4蛋白和膜表面蛋白的表达明显增加,0.1μmol/L达到高峰,干预不同的时间,12h开始增加,24h达到高峰,呈明显的浓度时间依赖性。Valsartan能抑制AngⅡ诱导的TLR4表达。ERK1/2和p38的抑制剂不能抑制AngⅡ诱导的TLR4表达。【结论】 1.血管紧张素Ⅱ能增加DC表面CD40的表达,但不是DC典型的成熟诱导剂。 2.血管紧张素Ⅱ通过AT1受体,ERK1/2、p38信号通路以及NF-κB活化诱导DC分泌IL-6和TNF-α,促进MCP-1和MIP-1α的表达。 3.血管紧张素Ⅱ可能通过AT2受体,ERK1/2、p38信号通路以及NF-κB活化,促进RANTES的表达。 4.血管紧张素Ⅱ增强LPS诱导的对异种T淋巴细胞的增殖反应,增强LPS诱导的细胞因子分泌和趋化因子的表达,提示血管紧张素Ⅱ能增强LPS诱导的DC免疫功能。 5.血管紧张素Ⅱ增强LPS诱导的DC免疫功能,可能通过AT1受体。 6.血管紧张素Ⅱ增强LPS诱导的ERK1/2活化,对p38的活化影响不大。 7.血管紧张素Ⅱ通过AT1受体上调DC表面TLR4的表达,这可能是血管紧张素Ⅱ增强LPS诱导的DC免疫功能的机制。
[Abstract]:[background and purpose]
AS is considered to be a chronic inflammatory process, thickening of the vascular wall lesions is the connective tissue for a proliferative reaction of inflammatory cytokines, various specific cell types, risk factors and exogenous antigen reaction. Dendritic cells are widely distributed in different tissues of the machine body antigen a cell, recently thought center DC may in AS immune reaction. A large number of studies found that the renin-angiotensin system may be involved in the occurrence and development of atherosclerosis. Basic research found Ang II AT1 receptor by stimulating the secretion of inflammatory mediators, induce cell apoptosis, promote ox-LDL uptake, oxygen free radicals and effect of fiber soluble functions involved in many aspects such as the process of atherosclerosis. The stimulate secretion of inflammatory mediators is one of the most important mechanisms, some researchers proposed Ang II special cytokines. Previous studies found that Ang II cells of plaque (endothelial cells, smooth muscle cells, macrophages) are proinflammatory mediators secretion: stimulate endothelial cell adhesion molecule expression and increase the adhesion of mononuclear cells to endothelium and promote plaque induced MMP-2 expression in endothelial cells, monocytes; oxygen free radicals or macrophages induced by induced vascular smooth muscle cell; the secretion of IL-6, MCP-1, and Ang II. Research on promoting migration of antigen presenting cells DC is less, its function and mechanism is not clear. Some studies reported that DC expression of angiotensin, angiotensin converting enzyme, angiotensin II receptor with mature change, suggesting that RAS system may promote the expression of DC cytokines.Ang II stimulated mononuclear cells to the differentiation of DC, ARB and losartan can block the monocyte to macrophage like cell differentiation as RAS this effect. The core of Ang II can be used as immune antigen induced AS induced maturation of DC, thereby enhancing the ability to activate T cells, promote plaque formation, there is no conclusion. If the immune Ang II can induce the maturation of DC, promote the secretion of inflammatory mediators, the mechanism of how? The purpose of this study is dendritic cells derived from mononuclear cells by in vitro, evaluate the Ang effect on DC immune function, and Study on the mechanism, to provide new ideas for the prevention and treatment of atherosclerosis.
[experimental method]
Isolation and culture of 1. cells: the separation of liquid from healthy adult peripheral white blood cell suspension mononuclear cells were isolated by lymphocyte, and then select CD14~+ beads with purity of over 98% CD14~+ mononuclear cells, including 20ng/ml rhGM-CSF, 10ng/ml rhIL-4 RPMI1640 medium, fifth days to collect cell suspension as immature DC (immature DC imDC, 100ng/ml, LPS) and cultured for 24 hours to collect up to mature DC (mature DC, mDC).
2. flow cytometry was used to determine the expression of surface molecules on DC.
3. mixed lymphocyte reaction was used to determine the ability of DC to promote lymphocyte proliferation.
The phagocytosis of DC was measured by the 4. phagocytosis test.
Cytokine in cell culture supernatant was measured by 5.ELISA.
6. semi quantitative RT-PCR was used to determine the expression of chemokine in dendritic cells.
7. Western blot was used to determine the expression of phosphorylated ERK1/2, p38 and TLR4.
8. the activity level of NF- kappa B in the nucleus was measured by EMSA method.
[results]
The expression level of CD40 in group 1.Ang was higher than that in control group. There was no significant difference in the expression of CD86, CD83 and HLA-DR. There was no difference between V+Ang group II and control group. There was no significant difference in the expression of cell surface molecules between Ang group II and control group under.LPS stimulation.
The ability of 2.Ang group II DC to phagocytosis and promote the proliferation of xenogeneic T lymphocytes was not significantly different from that of the control group. Compared with the control group, the proportion of T cells in the Ang group II group was higher than that in the control group under the stimulation of.LPS, but there was no difference between V+Ang group II and control group.
After 3.Ang II intervention, IL-6 and TNF- alpha in DC culture supernatant were significantly higher than those in the control group. Valsartan could inhibit IL-6 induced by angiotensin II and TNF- alpha secretion.LPS stimulation, and the TNF- alpha and the DC in DC culture supernatant of Ang group II were higher than those of the control group, and there was no difference between group II and control group.
The expression of DC, MCP-1, RANTES and MIP-1 alpha increased after 4.Ang II intervention. Valsartan inhibited the expression of MCP-1 and MIP-1 alpha induced by Ang II, but did not inhibit the expression of RANTES.
5.Ang II stimulates phosphorylation of ERK1/2, p38 expression, ERK1/2, p38 inhibitors can inhibit the secretion of IL-6, TNF- A and the expression of MCP-1, RANTES, MIP-1 alpha induced by Ang II.
6.Ang II can significantly increase the activation of DC NF- kappa B, and Valsartan can inhibit the activation of NF- kappa B induced by Ang II.
7.LPS stimulated the level of phosphorylated ERK1/2 in Ang II Group 5min and 15min was higher than that in the control group. Valsartan inhibited the ERK1/2 expression of Ang II enhanced phosphorylation. There was no significant difference between.Ang group II and the control group in the level of phosphorylation p38.
8. concentration of Ang II 24h 0.0001-1 mol/L intervention, the expression of TLR4 protein and membrane surface protein increased significantly, 0.1 mol/L peak, different intervention time, 12h began to increase, reached the peak at 24h, in a concentration time dependent.Valsartan significantly inhibited Ang II induced TLR4 expression of.ERK1/2 and p38 inhibitors no inhibition of Ang induced by TLR4.
[Conclusion]
1. angiotensin II can increase the expression of CD40 on the surface of DC, but it is not a typical mature inducer of DC.
2. angiotensin II promotes the expression of MCP-1 and MIP-1 alpha by inducing DC secretion of IL-6 and TNF- a through the activation of AT1 receptor, ERK1/2, p38 signaling pathway and NF- kappa B activation.
3. angiotensin II may promote the expression of RANTES through the activation of AT2 receptor, ERK1/2, p38 signaling pathway and NF- kappa B.
4. angiotensin II enhances LPS induced proliferation of xenogeneic T lymphocytes, enhances LPS induced cytokines secretion and expression of chemokines, suggesting that angiotensin II can enhance LPS induced DC immune function.
5. angiotensin II enhances the DC immune function induced by LPS and may pass through the AT1 receptor.
6. angiotensin II enhanced the activation of ERK1/2 induced by LPS, which had little effect on the activation of p38.
7. angiotensin II up-regulated the expression of TLR4 on the DC surface through the AT1 receptor, which may be the mechanism of angiotensin II enhancing the immune function of DC induced by LPS.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R392

【参考文献】