本文選題：動脈粥樣硬化　切入點：樹突狀細胞　出處：《浙江大學》2008年博士論文

【摘要】： 【背景和目的】 目前認為AS是一種慢性炎癥反應過程,增厚的病灶是血管壁內(nèi)結(jié)締組織對致炎因子的一種增生性反應,是各種特異的細胞、分子對外源性的危險因子與抗原的反應。樹突狀細胞是一種廣泛分布于機體各組織的抗原遞呈細胞,近來認為DC可能處于AS免疫反應的中心地位。大量的研究發(fā)現(xiàn)腎素-血管緊張素系統(tǒng)可能參與了動脈粥樣硬化的發(fā)生發(fā)展過程�；A(chǔ)研究發(fā)現(xiàn)AngⅡ通過AT1受體刺激炎癥介質(zhì)的分泌、誘導細胞凋亡、促進ox-LDL的攝取、產(chǎn)生氧自由基和影響纖溶功能等多方面參與動脈粥樣硬化過程。其中刺激炎癥介質(zhì)的分泌是最重要的機制之一,甚至有研究者提出AngⅡ為特殊的細胞因子。以往的研究發(fā)現(xiàn)AngⅡ?qū)Χ喾N斑塊內(nèi)的細胞成分(內(nèi)皮細胞、平滑肌細胞、巨噬細胞)均有促炎癥介質(zhì)分泌的作用:刺激內(nèi)皮細胞表達黏附分子及增加單核細胞對內(nèi)皮的黏附而促進斑塊的發(fā)展,誘導內(nèi)皮細胞表達MMP-2;誘導單核或巨噬細胞產(chǎn)生氧自由基;誘導平滑肌細胞分泌IL-6、MCP-1,促進遷移。而AngⅡ?qū)乖f呈細胞DC的研究則較少,其具體的作用及機制尚不清楚。有研究曾報道DC表達血管緊張素原、血管緊張素轉(zhuǎn)換酶、血管緊張素Ⅱ受體隨其成熟而改變,推測RAS系統(tǒng)可能促進DC表達炎癥因子。AngⅡ促進單核細胞向DC的分化,ARB氯沙坦能阻斷這種作用而使單核細胞向巨噬樣細胞分化。作為RAS系統(tǒng)中居于核心位置的AngⅡ能否作為致AS抗原物誘導DC的免疫成熟,從而增強激活T細胞的能力,促進斑塊的形成,目前尚無定論。如果AngⅡ能誘導DC的免疫成熟,促進炎癥介質(zhì)的分泌,其機制又如何?本研究的目的是利用體外培養(yǎng)人單核細胞源性的樹突狀細胞,評價AngⅡ?qū)C免疫功能的影響,并對其中的機制進行初探,為防治動脈粥樣硬化提供新的思路。 【實驗方法】 1.細胞的分離和培養(yǎng):用淋巴細胞分離液從健康成人外周血白細胞懸液分離單個核細胞,再用CD14~+磁珠分選出純度大于98%的CD14~+單核細胞,用含20ng/ml rhGM-CSF、10ng/ml rhIL-4的RPMI1640培養(yǎng)基培養(yǎng),第5天收集懸浮細胞作為未成熟DC(immature DC,imDC),加100ng/ml的LPS繼續(xù)培養(yǎng)24小時收集起來即為成熟DC(mature DC,mDC)。 2.流式細胞學測定DC表面分子的表達。 3.混合淋巴細胞反應測定DC的促淋巴細胞增殖的能力。 4.吞噬實驗測定DC的吞噬功能。 5.ELISA測定細胞培養(yǎng)上清中細胞因子。 6.半定量RT-PCR測定樹突狀細胞趨化因子的表達。 7.采用Western印跡測定磷酸化ERK1/2、p38及TLR4的表達。 8.采用EMSA法測定核內(nèi)NF-κB活性水平。 【結(jié)果】 1.AngⅡ組細胞表達CD40較對照組增高,CD86、CD83、HLA-DR的表達則無明顯差異,V+AngⅡ組CD40與對照組無差異。LPS刺激下,AngⅡ組與對照組細胞表面分子的表達無明顯差異。 2.AngⅡ組DC的吞噬、促異種T淋巴細胞增殖的能力與對照組相比無明顯差異。LPS刺激下,AngⅡ組子代T細胞比例高于對照組,V+AngⅡ組與對照組無差異。 3.AngⅡ干預后的DC培養(yǎng)上清中IL-6、TNF-α明顯高于對照組,Valsartan能抑制血管緊張素Ⅱ誘導的IL-6、TNF-α分泌。LPS刺激下,AngⅡ組DC培養(yǎng)上清中TNF-α和IL-12均高于對照組,V+AngⅡ組與對照組無差異。 4.AngⅡ干預后的DC表達MCP-1、RANTES、MIP-1α增加,Valsartan能抑制AngⅡ誘導的MCP-1、MIP-1α表達,但不抑制RANTES的表達。LPS刺激下,AngⅡ組DC MCP-1、RANTES、MIP-1α、IP-10的表達均高于對照組,V+AngⅡ組與對照組無差異。 5.AngⅡ刺激磷酸化ERK1/2、p38的表達,ERK1/2、p38的抑制劑能抑制AngⅡ誘導的IL-6、TNF-α的分泌以及MCP-1、RANTES、MIP-1α的表達。 6.AngⅡ可顯著增加DC NF-κB的活化,Valsartan能抑制AngⅡ誘導的NF-κB活化。 7.LPS刺激下,AngⅡ組5min、15min的磷酸化ERK1/2的水平高于對照組,Valsartan能抑制AngⅡ增強的磷酸化ERK1/2表達。AngⅡ組與對照組磷酸化p38的水平無明顯差異。 8.濃度為0.0001-1μmol/L的AngⅡ干預24h,TLR4蛋白和膜表面蛋白的表達明顯增加,0.1μmol/L達到高峰,干預不同的時間,12h開始增加,24h達到高峰,呈明顯的濃度時間依賴性。Valsartan能抑制AngⅡ誘導的TLR4表達。ERK1/2和p38的抑制劑不能抑制AngⅡ誘導的TLR4表達。 【結(jié)論】 1.血管緊張素Ⅱ能增加DC表面CD40的表達,但不是DC典型的成熟誘導劑。 2.血管緊張素Ⅱ通過AT1受體,ERK1/2、p38信號通路以及NF-κB活化誘導DC分泌IL-6和TNF-α,促進MCP-1和MIP-1α的表達。 3.血管緊張素Ⅱ可能通過AT2受體,ERK1/2、p38信號通路以及NF-κB活化,促進RANTES的表達。 4.血管緊張素Ⅱ增強LPS誘導的對異種T淋巴細胞的增殖反應,增強LPS誘導的細胞因子分泌和趨化因子的表達,提示血管緊張素Ⅱ能增強LPS誘導的DC免疫功能。 5.血管緊張素Ⅱ增強LPS誘導的DC免疫功能,可能通過AT1受體。 6.血管緊張素Ⅱ增強LPS誘導的ERK1/2活化,對p38的活化影響不大。 7.血管緊張素Ⅱ通過AT1受體上調(diào)DC表面TLR4的表達,這可能是血管緊張素Ⅱ增強LPS誘導的DC免疫功能的機制。
[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.

【學位授予單位】：浙江大學
【學位級別】：博士
【學位授予年份】：2008
【分類號】：R392

【參考文獻】

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1 姚康;葛均波;孫愛軍;洪曉武;施鴻毓;黃榕，

本文編號：1676162