C反应蛋白和脂蛋白对人外周血单核细胞趋化蛋白-1及其受体表达的影响
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摘要
研究背景
冠心病已是当今严重危害人类健康、影响人们生活质量的最常见心血管疾病之一。冠心病的发病机制尚不十分清楚,研究与冠心病相关的危险因素尤为重要。来自Framingham Heart Study等研究的资料已经证实,高血压、糖尿病、吸烟、低密度脂蛋白升高、高密度脂蛋白降低、年龄和性别是冠心病的独立危险因素和心血管事件发生的独立预测因子。近年人们又提出冠心病的发生发展机制与炎症反应密不可分。本课题就是基于如上所述,以冠心病发生的炎症机制为切入点,研究经典危险因素血脂异常、C反应蛋白与炎症反应之间的关系,阐明冠心病发病机制之一,从而丰富治疗冠心病的理论依据。
动脉粥样硬化(atherosclerosis,AS)是冠心病的病理基础,“脂质沉积”学说是AS发病机制的经典假说。自从Ross等提出了AS的“损伤反应”学说后,越来越多的研究提示AS是一种血管受损后的炎症过程。“脂质沉积”学说与“炎症反应”学说并非互相排斥,而是密切联系的。目前认为AS的最早变化是动脉内膜的损伤、单核-巨噬细胞内膜下沉积以及低密度脂蛋白(Low density lipoprotein,LDL)的氧化修饰。氧化低密度脂蛋白(Oxidized low density lipoprotein,Ox-LDL)和自由基的毒性作用可以破坏血管内膜完整性,损伤血管内皮细胞,并且促使内皮细胞分泌单核细胞刺激因子和单核细胞趋化蛋白-1(monocyte Chemoattractantprotein-1,MCP-1),此外还可以使一些细胞成分活化,诱导肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-6(interleukin-6,IL-6)、粘附分子如血管细胞黏附分子-1(VCAM-1)、细胞间黏附分子-1(ICAM-1)和其他炎性介质的表达。MCP-1吸引血液中的单核细胞向受损血管内皮区域聚集,VCAM-1介导单核细胞粘附至受损血管内膜,继而分化为巨噬细胞并分泌炎症因子,其中包括了MCP-1、ICAM-1,吸引更多单核-巨噬细胞向内皮受损区域集中、粘附,增加炎症细胞浸润。局部聚集的巨噬细胞吞噬脂质,形成泡沫细胞,造成动脉粥样硬化。所以,血清中MCP-1含量可以一定程度上反映体内炎症活动水平。趋化因子受体2(C-C chemokine receptor 2,CCR2)是MCP-1的特异性受体,广泛分布于单核细胞、内皮细胞和平滑肌细胞表面。MCP-1/CCR2一起参与了许多炎症相关疾病的发生和发展。研究证实Ox-LDL可通过血凝素氧化低密度脂蛋白受体-1(LOX-1)促进内皮细胞和单核细胞MCP-1/CCR2的表达,同时也有研究认为Ox-LDL并不能上调MCP-1/CCR2通路。
C反应蛋白(C-reactive protein,CRP)是体内炎症和组织损伤的敏感性指标。大量研究结果表明CRP也是AS的独立危险因素。美国心脏病学会(AHA)推荐高敏CRP(high sensitivity C-reactive protein,hs-CRP)水平为评价冠心病人群危险度分层的依据之一,其中CRP≥3mg/L为中危人群的界值,CRP≥10mg/L为高危人群的标准。CRP超过20mg/L需要考虑有无感染等其他非冠心病因存在。也有研究发现CRP可以上调低密度脂蛋白受体LOX-1表达,增加细胞内胆固醇蓄积;促进血管平滑肌细胞凋亡,增加斑块不稳定性;通过核因子κB途径诱导VCAM-1基因表达,促进单核细胞在血管损伤区域的聚集、黏附和浸润;抑制内皮细胞一氧化氮的合成和血管内膜损伤的修复。因此有研究人员认为CRP不仅是炎症反应的标志物和预测分子,还有可能直接参与了AS的形成。由于CRP的升高受到众多生理和病理因素的影响,因此缺乏特异性和敏感性。CRP究竟是不是冠心病的独立危险因素?CRP与冠心病的相关性如何?CRP是不是冠心病或AS的致病因素?上述问题仍然没有充分的证据给予肯定的回答。CRP与冠心病发生发展的关系仍是当前研究的一个热点。而CRP与MCP-1及其受体CCR2关系的研究报道较少。
高密度脂蛋白(High density lipoprotein,HDL)是目前所知与AS呈负相关的脂蛋白。目前认为HDL抗AS的作用归因于其参与了胆固醇逆向转运。细胞内游离胆固醇和磷脂经ATP结合盒转运子A1(ATP binding cassette transporterA1,ABCA1)转运至细胞外,与载脂蛋白AⅠ(apoAⅠ)结合组装为新生HDL,不断接受由周围细胞内移出的游离胆固醇逐渐形成成熟HDL,HDL将胆固醇酯转移至LDL等,通过LDL受体进入肝脏代谢。也有研究发现HDL除了调节胆固醇逆转运外,还可以抑制黏附分子VCAM-1、ICAM-1的合成以及核转录因子κB(NF-κB)的转录活性。因此有研究人员认为,HDL具有抑制炎症反应的作用继而抑制甚至逆转冠心病发生。同样,没有确切的证据可以证实HDL的这种作用。HDL是否可以通过减少MCP-1的含量发挥其抗AS作用则尚未见报道。
鉴于上述证据,我们可以假设Ox-LDL和CRP可以通过刺激单核细胞合成分泌MCP-1和表达MCP-1受体促进AS发病早期单核细胞的聚集和活化;HDL可能通过抑制MCP-1及其受体的表达而发挥对心血管系统的保护作用,希望通过该研究可以丰富防治冠心病的理论基础。
目的
1.观察在不同浓度氧化低密度脂蛋白(Ox-LDL)、和不同浓度C反应蛋白(CRP)刺激下人单核细胞MCP-1及其受体CCR2基因表达水平和MCP-1蛋白含量的变化情况,以明确CRP和Ox-LDL是否可以通过上调MCP-1/CCR2基因和蛋白表达来促进单核细胞聚集;
2.高密度脂蛋白(HDL)是否可以抑制CRP和Ox-LDL的此种作用,探讨HDL抗AS作用的可能机制。
3.探讨Ox-LDL、CRP、HDL与单核细胞MCP-1及CCR2表达之间可能存在的联系以及在动脉粥样硬化形成中的作用。
对象和方法
1.对象
体外培养的人外周血来源的单核细胞。
2.方法
2.1 Ox-LDL的制备
采用改良的一次性密度梯度超速离心法从健康人新鲜血液分离出LDL。将最终浓度调整至500mg/L。采用Cu2+诱导法对LDL进行氧化,在浓度为100μmol/L的EDTA溶液中终止氧化。Bradford法测定蛋白含量。
2.2单核细胞的分离和培养
常规密度梯度离心法从人外周血分离获得单个核细胞,加入含10%胎牛血清的RPMI-1640培养基,以5×10~5/mL的密度接种于24孔培养板中,置饱和湿度、体积分数为0.05的CO2 37℃孵育箱中培养3天,加入不同浓度的Ox-LDL、CRP进行刺激,加入HDL观察其对Ox-LDL、CRP的抑制作用。
2.3实验分组每组3个复孔
2.3.1 Ox-LDL和CRP对单核细胞MCP-1和CCR2基因表达的影响
(1)对照组加入含磷酸盐缓冲液(PBS)的等量1640培养基培养单核细胞;
(2)不同浓度Ox-LDL组:终浓度分别为20mg/L、40mg/L、80mg/L的Ox-LDL与单核细胞孵育48小时;
(3)不同浓度CRP组:浓度分别为3mg/L、10mg/L、20mg/L的CRP与单核细胞孵育48小时;
(4)提取细胞总RNA行RT-PCR检测,每个培养孔提取2次细胞RNA送检,即n=6。
2.3.2 Ox-LDL和CRP对单核细胞MCP-1蛋白表达的影响
(1)对照组:加入含磷酸盐缓冲液(PBS)的等量1640培养基培养单核细胞作为对照组;
(2)不同浓度Ox-LDL组:终浓度分别为20mg/L、40mg/L、80mg/L的Ox-LDL与单核细胞孵育48小时;
(3)不同浓度CRP组:浓度分别为3mg/L、10mg/L、20mg/L的CRP与单核细胞孵育48小时;
(4)收集细胞上清进行ELISA检测,每个培养孔提取3次细胞上清送检,即n=9。
2.3.3 HDL对单核细胞MCP-1和CCR2基因表达以及MCP-1蛋白表达的影响
(1)对照组:加入含磷酸盐缓冲液(PBS)的等量1640培养基培养单核细胞作为对照组;
(2)HDL组:加入浓度为500mg/L HDL及1640培养基,提取细胞总RNA行RT-PCR检测,每个培养孔提取2次细胞RNA送检,即n=6;收集细胞上清进行ELISA检测,每个培养孔提取3次细胞上清送检,即n=9;
2.3.4 HDL对Ox-LDL、CRP介导单核细胞MCP-1和CCR2基因表达的影响
(1)不同浓度Ox-LDL对照组:终浓度分别为20mg/L、40mg/L、80mg/L的Ox-LDL与单核细胞孵育48小时;
(2)不同浓度CRP对照组:浓度分别为3mg/L、10mg/L、20mg/L的CRP与单核细胞孵育48小时;
(3)不同浓度Ox-LDL与HDL共处理组:分别用500mg/L HDL+20mg/LOx-LDL、500mg/L HDL+40mg/L Ox-LDL、500mg/L HDL+80mg/L Ox-LDL与单核细胞孵育48小时;
(4)不同浓度CRP组与HDL共处理组:分别用500mg/L HDL+3mg/L CRP、500mg/L HDL+10mg/L CRP、500mg/L HDL+20mg/L CRP与单核细胞孵育48小时;
(5)提取细胞总RNA行RT-PCR检测,每个培养孔提取2次细胞RNA送检,即n=6。
2.3.5 HDL对Ox-LDL、CRP介导单核细胞MCP-1蛋白表达的干预作用
(1)不同浓度Ox-LDL组:终浓度分别为20mg/L、40mg/L、80mg/L的Ox-LDL与单核细胞孵育48小时;
(2)不同浓度CRP组:浓度分别为3mg/L、10mg/L、20mg/L的CRP与单核细胞孵育48小时;
(3)不同浓度Ox-LDL与HDL共处理组:分别用500mg/L HDL+20mg/LOx-LDL、500mg/L HDL+40mg/L Ox-LDL、500mg/L HDL+80mg/L Ox-LDL与单核细胞孵育48小时;
(4)不同浓度CRP组与HDL共处理组:分别用500mg/L HDL+3mg/L CRP、500mg/L HDL+10mg/L CRP、500mg/L HDL+20mg/L CRP与单核细胞孵育48小时:
(5)收集细胞上清进行ELISA检测,每个培养孔提取3次细胞上清送检,即n=9。
2.4.实时荧光定量RT-PCR法检测MCP-1和CCR2 mRNA表达
提取细胞样品总RNA,设计内参β-actin和目的基因引物,采用RT-PCR扩增,同时加入荧光染料,通过检测目的基因与内参基因达到荧光设定值所需循环数(Ct值)(ΔCt)并对不同处理组目的基因的ΔCt进行比较,可对未知标本靶基因的原始拷贝数做出判断。
2.5酶联免疫吸附试验(Enzyme-Linked ImmunosorbentAssays,ELISA)检测细胞培养液MCP-1蛋白含量,采用双抗体夹心ELISA法。
3.统计学方法
RT-PCR结果(ΔCt)和ELISA结果(ug/L)以均数±标准差表示,进行方差齐性检验。方差齐(P＞0.10)则采用方差分析,方差不齐则采用Tamhane法校正。若方差分析结果提示各组间差异具有显著性,则用Bonferroni法进行组间多重比较。采用单因素方差分析分别对不同浓度Ox-LDL和不同浓度CRP的作用进行统计学处理;采用两样本t检验对HDL对单核细胞MCP-1基因、蛋白和CCR2基因表达进行统计学分析;采用两因素方差分析对HDL的干预作用进行统计学处理。所有统计学处理均采用SPSS 13.0统计软件进行分析。P＜0.05表明有统计学差异。
结果
1.不同浓度Ox-LDL均可以促进单核细胞MCP-1 mRNA的表达(F=88.90,P=0.000);与对照组相比,20mg/L、40mg/L、80mg/L Ox-LDL处理组MCP-1基因表达上调了1.97倍、3.16倍和4.07倍;组间多重比较结果显示,20mg/L与40mg/L、40mg/L与80mg/L差异具有统计学意义(P=0.000,P=0.007),即Ox-LDL促进MCP-1基因表达的作用存在浓度效应。
2.不同浓度Ox-LDL均可促进单核细胞CCR2 mRNA的表达(F=129.25,P=0.000);与对照组相比,20mg/L、40mg/L、80mg/L Ox-LDL处理组MCP-1基因表达上调了2.12倍、2.54倍和3.02倍;组间多重比较结果显示,20mg/L与40mg/L、40mg/L与80mg/L差异具有统计学意义(P=0.008,P=0.009),Ox-LDL促进CCR2基因表达的作用存在浓度效应。
3.3mg/L CRP对MCP-1 mRNA表达的影响不具有统计学意义,10mg/L、20mg/L CRP则可以促进单核细胞MCP-1 mRNA的表达(F=122.07,P=0.000);与对照组相比,10mg/L、20mg/L CRP处理组MCP-1基因表达上调了3.09倍和4.04倍;10mg/L与20mg/L之间差异具有统计学意义(P=0.002),即CR2促进MCP-1基因表达的作用存在一定的浓度效应。
4.3mg/L CRP对CCR2 mRNA表达的影响也不具有统计学意义,而10mg/L、20mg/L CRP则可以促进单核细胞CCR2 mRNA的表达(F=177.35,P=0.000);与对照组相比,10mg/L、20mg/L CRP处理组MCP-1基因表达上调了2.96倍和3.59倍;10mg/L与20mg/L之间差异具有统计学意义(P=0.015),即CRP促进CCR2基因表达的作用存在一定的浓度效应。
5.500mg/L的HDL对单核细胞MCP-1(t=0.01,P=0.992)和CCR2基因(t=-0.623,P=0.547)表达的影响没有统计学意义;
6.500mg/L HDL可以抑制Ox-LDL促进MCP-1和CCR2 mRNA表达的作用(F=36.02,P=0.000);20mg/L Ox-LDL+500mg/L HDL组MCP-1和CCR2 mRNA表达量为20mg/L Ox-LDL的57.2%和49.5%;40mg/L Ox-LDL+500mg/L HDL组MCP-1和CCR2 mRNA表达量为40mg/L Ox-LDL的77.0%和86.7%;80mg/LOx-LDL+500mg/L HDL组MCP-1和CCR2 mRNA表达量为80mg/L Ox-LDL的71.9%和85.6%:
7.500mg/L HDL可以抑制CRP对单核细胞MCP-1和CCR2 mRNA表达的促进作用(F=20.4 1,P=0.000);10mg/L CRP+500mg/L HDL组MCP-1和CCR2 mRNA表达量为10mg/L CRP的73.9%和82.8%;20mg/L CRP+500mg/L HDL组MCP-1和CCR2 mRNA表达量为20mg/L CRP的69.8%和83.2%:
8.各浓度的Ox-LDL均可以促进单核细胞产生MCP-1(F=476.90,P=0.000);20mg/L与40mg/L、40mg/L与80mg/L差异具有统计学意义(P=0.000);
9.10mg/L、20mg/L CRP均可以促进单核细胞产生MCP.1(F=152.19.P=0.000);3mg/L CRP对单核细胞MCP-1蛋白含量的影响没有统计学意义(P=0.203);10mg/L与20mg/L CRP差异具有统计学意义(P=0.000);
10.500mg/L HDL对单核细胞MCP-1蛋白表达的影响没有统计学意义(t=-0.585,p=0.567);
11.500mg/L HDL可抑制Ox-LDL促进MCP-1蛋白表达的作用(F=14.12,P=0.000);
12.500mg/L HDL可抑制CRP促进MCP-1蛋白表达的作用(F=10.88,P=0.002)。
结论
1.在体外,Ox-LDL和C反应蛋白均可以促进单核细胞MCP-1和CCR2 mRNA的表达和MCP-1的分泌,对炎症反应起着重要的推动作用;
2.HDL对单核细胞MCP-1/CCR2没有直接作用,但可以抑制Ox-LDL、CRP上调MCP-1和CCR2基因表达的作用,这可能是HDL与AS发病呈负相关的机制之一;
3.CRP、Ox-LDL和HDL对MCP-1作用的信号转导通路需要进一步研究。
Background
As one of the most common cardiovascular diseases,Coronary heart disease (CHD) has been seriously jeopardizing health and quality of life of human beings. The pathogenesis of CHD,However,is not thoroughly identified.It is of much importance to study the relevant risk factors of CHD.Data from several studies,such as the Framingham Heart Study,have demonstrated some independent risk factors of CHD and predictors for cardiovascular outcomes,such as elevated blood pressure, diabetes mellitus,smoking,high low-density lipoprotein,reduced high-density lipoprotein,age and gender.Recently,some researchers believe that inflammation is involved in the pathogenesis and progress of CHD.So we have paid much attention on the inflammatory mechanism of CHD and try to illustrate pathogenesis of CHD in terms of relationships among traditional risk factors such as lipid,C-reactive protein (CRP) and inflammation.
Atherosclerosis(AS) is pathological basis of CHD.Since Ross raised the response-to-injury theory of AS in 1999,increasing evidence suggests AS is an inflammatory process after vascular injury.Endothelial injury and oxidative modification of low-density lipoprotein(LDL) are considered the earliest stage of AS pathogenesis.Low-density lipoprotein(LDL),which may be modified by oxidation, glycation(in diabetes),aggregation,association with proteoglycans,or incorporation into immune complexes,is a major cause of injury to the endothelium and underlying smooth muscle.When LDL particles become trapped in an artery,they can undergo progressive oxidation and be internalized by macrophages by means of the scavenger receptors on the surfaces of these cells.The internalization leads to the formation of lipid peroxides and facilitates the accumulation of cholesterol esters,resulting in the formation of foam cells.Oxidized-LDL(Ox-LDL) is chemotactic for other monocytes and can up-regulate the expression of genes for macrophage colony-stimulating factor and monocyte chemoattractant protein-1(MCP-1) derived from endothelial cells,and other inflammatory cytokines such as tumor necrosis factor-α(TNF-α), interleukin-6(IL-6),vascular cell adhesion molecule-1(VCAM-1) and intercellular cell adhesion molecule-1(ICAM-1).These cytokines may help expand the inflammatory response by stimulating the replication and secretion of monocyte-derived macrophages and the entry of new monocytes into lesions.Those activated macrophages have enhanced secretion property leading to increased level of MCP-1,ICAM-1 and VCAM-1.Such a positive-feedback results in the aggregation and adhesion to injury endothelium of monocyte-macrophages.These macrophages transformed into foam cells and,thus,led to the AS.So,serum level of MCP-1 could partly reflect the extent and amplitude of inflammation.The C-C chemokine receptor 2,commonly known as CCR2,is the specific receptor for MCP-1 and widely expression on endothelial cells,monocytes and smooth muscle cells.MCP-1/CCR2 plays an important role in pathogenesis and progress of inflammation relevant diseases,including AS.
C-reactive protein(CRP) is a sensitive marker for inflammation and tissue injury. A large number of research indicated that CRP was an independent risk factor for AS. The American heart Association(AHA) recommended the use of CRP as a prognostic marker in patients with acute coronary syndrome(ACS) in addition to other prognostic factors,including troponin levels.An evaluation of CRP levels at the time of admission should be included in the evaluation of the patient risk profile,including clinical data,associated diseases,markers of myocardial necrosis,left ventricle performance,and age.A cutoff level of 10mg/L was identified as marker of higher risk for death and possibly myocardial infarction in ACS,whereas a cutoff of 3 mg/L identifies a group of patients at intermediate risk and a high rate of recurrent events.
Other researches indicated that CRP enhanced gene expression of Lectin- like oxidized low density lipoprotein receptor—1(LOX—1) leading to intracellular accumulation of cholesterol,induced apoptosis of vascular smooth muscle cell and VCAM-1 gene expression through nuclear factor-κB pathway.CRP was also found to attenuate nitric oxide synthesis in endothelial cell and repair in injured endothelium. These findings provided direct evidence for the hypothesis that CRP may not only a marker and predictor,but a direct cause for AS through upregulate inflammatory response.However,little is known about the relationship between CRP and MCP-1.
The results of numerous epidemiological studies have established that high density lipoprotein(HDL) levels correlate inversely with the risk of developing cardiovascular disease.This relationship reflects several functions of HDL,the most extensively studied of which is their ability to remove excess cholesterol from cells, such as macrophages in the artery wall,in the first step of the reverse cholesterol transport pathway.In recent years it has become increasingly apparent that HDL also has potent anti-inflammatory properties.This has been demonstrated in vitro as well as in vivo.For example,the cytokine-induced expression of VCAM-1,ICAM-1,and E-selectin in cultured human umbilical vein endothelial cells(HUVECs) is inhibited by HDL in a concentration-dependent manner.It remains uncertain that whether HDL attenuates MCP-1 expression.
In summary,we hypothesized that CRP could enhance MCP-1 and CCR2 gene expression,which result in the aggregation and activation of monocytes in early stage of AS.HDL has potent anti-inflammatory properties through inhibiting MCP-1 and CCR2 expression.
Objectives:
1.To determine the effects of Ox-LDL and CRP on gene and protein expression of MCP-1 and CCR2 in monocytes.
2.To investigate whether HDL could regulate gene and protein expression of MCP-1 and CCR2 in monocytes which was elicited by Ox-LDL and CRP.
Methods:
1.Preparation of Ox-LDL
We separated and quantified the LDL from blood of healthy individuals by density gradient ultracentrifugation.Adjusted LDL to a final concentration of 500mg/L,then incubated it with 10mmol/L CuSO_4.The oxidation was ceased by adding 100μmol/L EDTA.Finally,Bradford method was utilized to detect the protein level of Ox-LDL.
2.Isolation and culture of monocytes
Monocytes were isolated from human peripheral blood by Ficoll-Hypaque density gradient centrifugation,inoculated in RPMI-1640 medium supplemented with 1%Penicillin and 10%fetal bovine serum(FBS) and incubated under the condition of 5%CO2,37.5℃.
3.Groups and cultural condition
(1)Monocytes were cultured with equal RPMI-1640 medium as control.
(2)Different concentration groups of Ox-LDL:Monocytes were treated with Ox-LDL (20mg/L、40mg/L and 80mg/L) for 48h,respectively.
(3)Different concentration groups of CRP:Monocytes were treated with CRP (3mg/L、10mg/L、20mg/L) for 48h,respectively.
(4)500mg/L HDL was added alone and cultured with monocyte to determine its role on both genes expression and MCP-1 protein level;
(5)Different concentration groups of Ox-LDL and CRP as described in(2) and(3), each group added 500mg/L HDL before culture.
4.Real-time quantitative PCR for MCP-1 and CCR2
MCP-1 and CCR2 mRNA was examined by Fluorescent quantitative RT-PCR. Total RNA extracted from cultured cells was reverse-transcripted and amplified using specific primers for human MCP-1 and CCR2.The 2(-Delta Delta C(T)) method is utilized to analyze the relative changes in gene expression from real-time quantitative PCR experiments.
5.ELISA assay for MCP-1 protein level
This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for MCP-1 has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any MCP-1 present is bound by the immobilized antibody.An enzyme-linked monoclonal antibody specific for MCP-1 is added to the wells.Following a wash to remove any unbound antibody-enzyme reagent,a substrate solution is added to the wells and color develops in proportion to the amount of MCP-1 bound in the initial step.The color development is stopped and the intensity of the color is measured.
6.Data analysis
SPSS 13.0 statistical software was used to analyze data.Data are presented as Mean±S.D.Statistical significance was determined in a multiple comparisons among different groups of data in which one-way ANOVA test indicated the presence of significant differences.P value＜0.05 was considered to be significant.
Results:
1.Effects of Ox-LDL on MCP-1 and CCR2
Incubation of monocytes with Ox-LDL of different concentration for 48h significantly enhanced the expression of MCP-1(mRNA and protein) and CCR2 mRNA in a concentration-dependent manner(P=0.000).When compared with control,MCP-1 mRNA expression increased to 1.97,3.16 and 4.07 fold and protein expression increased(P=0.000) while CCR2 mRNA expression increased to 2.12, 2.54 and 3.02 fold(P=0.009) after treated with Ox-LDL of different concentrations.
2.Effects of CRP on MCP-1 and CCR2
Incubation of monocytes with CRP of different concentration for 48h significantly enhanced the expression of MCP-1(mRNA and protein) and CCR2 mRNA in a concentration-dependent manner(P=0.000).There was no significance between 3mg/L CRP group and control MCP-1 mRNA expression increased to 3.09 and 4.04 fold(P=0.000) and protein expression elevated(P=0.000) while CCR2 mRNA expression increased to 2.96 and 3.59 fold(P=0.005) after treated with CRP of different concentrations(10mg/L and 20mg/L),as compared with control.
3.HDL had no direct influence on on both genes expression and MCP-1 protein level;
4.Role of HDL in expression of MCP-1 and CCR2 elicited by Ox-LDL and CRP
Co-incubation of monocytes with HDL significantly inhibited Ox-LDL-induced and CRP-induced expression of MCP-1(mRNA and protein) and CCR2 mRNA.
Conclusions:
1.Oxidized low-density lipoprotein upregulates the mRNA and protein expression of MCP-1 and mRNA expression of CCR2;
2.C-reactive protein upregulates the mRNA and protein expression of MCP-1 and mRNA expression of CCR2;
3.High density lipoprotein which inhibits Ox-LDL-induced and CRP-induced upregulation of MCP-1 and CCR2 may exert beneficial effect in the prevention and treatment of atherosclerosis.
冠心病已是当今严重危害人类健康、影响人们生活质量的最常见心血管疾病之一。冠心病的发病机制尚不十分清楚,研究与冠心病相关的危险因素尤为重要。来自Framingham Heart Study等研究的资料已经证实,高血压、糖尿病、吸烟、低密度脂蛋白升高、高密度脂蛋白降低、年龄和性别是冠心病的独立危险因素和心血管事件发生的独立预测因子。近年人们又提出冠心病的发生发展机制与炎症反应密不可分。本课题就是基于如上所述,以冠心病发生的炎症机制为切入点,研究经典危险因素血脂异常、C反应蛋白与炎症反应之间的关系,阐明冠心病发病机制之一,从而丰富治疗冠心病的理论依据。
动脉粥样硬化(atherosclerosis,AS)是冠心病的病理基础,“脂质沉积”学说是AS发病机制的经典假说。自从Ross等提出了AS的“损伤反应”学说后,越来越多的研究提示AS是一种血管受损后的炎症过程。“脂质沉积”学说与“炎症反应”学说并非互相排斥,而是密切联系的。目前认为AS的最早变化是动脉内膜的损伤、单核-巨噬细胞内膜下沉积以及低密度脂蛋白(Low density lipoprotein,LDL)的氧化修饰。氧化低密度脂蛋白(Oxidized low density lipoprotein,Ox-LDL)和自由基的毒性作用可以破坏血管内膜完整性,损伤血管内皮细胞,并且促使内皮细胞分泌单核细胞刺激因子和单核细胞趋化蛋白-1(monocyte Chemoattractantprotein-1,MCP-1),此外还可以使一些细胞成分活化,诱导肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-6(interleukin-6,IL-6)、粘附分子如血管细胞黏附分子-1(VCAM-1)、细胞间黏附分子-1(ICAM-1)和其他炎性介质的表达。MCP-1吸引血液中的单核细胞向受损血管内皮区域聚集,VCAM-1介导单核细胞粘附至受损血管内膜,继而分化为巨噬细胞并分泌炎症因子,其中包括了MCP-1、ICAM-1,吸引更多单核-巨噬细胞向内皮受损区域集中、粘附,增加炎症细胞浸润。局部聚集的巨噬细胞吞噬脂质,形成泡沫细胞,造成动脉粥样硬化。所以,血清中MCP-1含量可以一定程度上反映体内炎症活动水平。趋化因子受体2(C-C chemokine receptor 2,CCR2)是MCP-1的特异性受体,广泛分布于单核细胞、内皮细胞和平滑肌细胞表面。MCP-1/CCR2一起参与了许多炎症相关疾病的发生和发展。研究证实Ox-LDL可通过血凝素氧化低密度脂蛋白受体-1(LOX-1)促进内皮细胞和单核细胞MCP-1/CCR2的表达,同时也有研究认为Ox-LDL并不能上调MCP-1/CCR2通路。
C反应蛋白(C-reactive protein,CRP)是体内炎症和组织损伤的敏感性指标。大量研究结果表明CRP也是AS的独立危险因素。美国心脏病学会(AHA)推荐高敏CRP(high sensitivity C-reactive protein,hs-CRP)水平为评价冠心病人群危险度分层的依据之一,其中CRP≥3mg/L为中危人群的界值,CRP≥10mg/L为高危人群的标准。CRP超过20mg/L需要考虑有无感染等其他非冠心病因存在。也有研究发现CRP可以上调低密度脂蛋白受体LOX-1表达,增加细胞内胆固醇蓄积;促进血管平滑肌细胞凋亡,增加斑块不稳定性;通过核因子κB途径诱导VCAM-1基因表达,促进单核细胞在血管损伤区域的聚集、黏附和浸润;抑制内皮细胞一氧化氮的合成和血管内膜损伤的修复。因此有研究人员认为CRP不仅是炎症反应的标志物和预测分子,还有可能直接参与了AS的形成。由于CRP的升高受到众多生理和病理因素的影响,因此缺乏特异性和敏感性。CRP究竟是不是冠心病的独立危险因素?CRP与冠心病的相关性如何?CRP是不是冠心病或AS的致病因素?上述问题仍然没有充分的证据给予肯定的回答。CRP与冠心病发生发展的关系仍是当前研究的一个热点。而CRP与MCP-1及其受体CCR2关系的研究报道较少。
高密度脂蛋白(High density lipoprotein,HDL)是目前所知与AS呈负相关的脂蛋白。目前认为HDL抗AS的作用归因于其参与了胆固醇逆向转运。细胞内游离胆固醇和磷脂经ATP结合盒转运子A1(ATP binding cassette transporterA1,ABCA1)转运至细胞外,与载脂蛋白AⅠ(apoAⅠ)结合组装为新生HDL,不断接受由周围细胞内移出的游离胆固醇逐渐形成成熟HDL,HDL将胆固醇酯转移至LDL等,通过LDL受体进入肝脏代谢。也有研究发现HDL除了调节胆固醇逆转运外,还可以抑制黏附分子VCAM-1、ICAM-1的合成以及核转录因子κB(NF-κB)的转录活性。因此有研究人员认为,HDL具有抑制炎症反应的作用继而抑制甚至逆转冠心病发生。同样,没有确切的证据可以证实HDL的这种作用。HDL是否可以通过减少MCP-1的含量发挥其抗AS作用则尚未见报道。
鉴于上述证据,我们可以假设Ox-LDL和CRP可以通过刺激单核细胞合成分泌MCP-1和表达MCP-1受体促进AS发病早期单核细胞的聚集和活化;HDL可能通过抑制MCP-1及其受体的表达而发挥对心血管系统的保护作用,希望通过该研究可以丰富防治冠心病的理论基础。
目的
1.观察在不同浓度氧化低密度脂蛋白(Ox-LDL)、和不同浓度C反应蛋白(CRP)刺激下人单核细胞MCP-1及其受体CCR2基因表达水平和MCP-1蛋白含量的变化情况,以明确CRP和Ox-LDL是否可以通过上调MCP-1/CCR2基因和蛋白表达来促进单核细胞聚集;
2.高密度脂蛋白(HDL)是否可以抑制CRP和Ox-LDL的此种作用,探讨HDL抗AS作用的可能机制。
3.探讨Ox-LDL、CRP、HDL与单核细胞MCP-1及CCR2表达之间可能存在的联系以及在动脉粥样硬化形成中的作用。
对象和方法
1.对象
体外培养的人外周血来源的单核细胞。
2.方法
2.1 Ox-LDL的制备
采用改良的一次性密度梯度超速离心法从健康人新鲜血液分离出LDL。将最终浓度调整至500mg/L。采用Cu2+诱导法对LDL进行氧化,在浓度为100μmol/L的EDTA溶液中终止氧化。Bradford法测定蛋白含量。
2.2单核细胞的分离和培养
常规密度梯度离心法从人外周血分离获得单个核细胞,加入含10%胎牛血清的RPMI-1640培养基,以5×10~5/mL的密度接种于24孔培养板中,置饱和湿度、体积分数为0.05的CO2 37℃孵育箱中培养3天,加入不同浓度的Ox-LDL、CRP进行刺激,加入HDL观察其对Ox-LDL、CRP的抑制作用。
2.3实验分组每组3个复孔
2.3.1 Ox-LDL和CRP对单核细胞MCP-1和CCR2基因表达的影响
(1)对照组加入含磷酸盐缓冲液(PBS)的等量1640培养基培养单核细胞;
(2)不同浓度Ox-LDL组:终浓度分别为20mg/L、40mg/L、80mg/L的Ox-LDL与单核细胞孵育48小时;
(3)不同浓度CRP组:浓度分别为3mg/L、10mg/L、20mg/L的CRP与单核细胞孵育48小时;
(4)提取细胞总RNA行RT-PCR检测,每个培养孔提取2次细胞RNA送检,即n=6。
2.3.2 Ox-LDL和CRP对单核细胞MCP-1蛋白表达的影响
(1)对照组:加入含磷酸盐缓冲液(PBS)的等量1640培养基培养单核细胞作为对照组;
(2)不同浓度Ox-LDL组:终浓度分别为20mg/L、40mg/L、80mg/L的Ox-LDL与单核细胞孵育48小时;
(3)不同浓度CRP组:浓度分别为3mg/L、10mg/L、20mg/L的CRP与单核细胞孵育48小时;
(4)收集细胞上清进行ELISA检测,每个培养孔提取3次细胞上清送检,即n=9。
2.3.3 HDL对单核细胞MCP-1和CCR2基因表达以及MCP-1蛋白表达的影响
(1)对照组:加入含磷酸盐缓冲液(PBS)的等量1640培养基培养单核细胞作为对照组;
(2)HDL组:加入浓度为500mg/L HDL及1640培养基,提取细胞总RNA行RT-PCR检测,每个培养孔提取2次细胞RNA送检,即n=6;收集细胞上清进行ELISA检测,每个培养孔提取3次细胞上清送检,即n=9;
2.3.4 HDL对Ox-LDL、CRP介导单核细胞MCP-1和CCR2基因表达的影响
(1)不同浓度Ox-LDL对照组:终浓度分别为20mg/L、40mg/L、80mg/L的Ox-LDL与单核细胞孵育48小时;
(2)不同浓度CRP对照组:浓度分别为3mg/L、10mg/L、20mg/L的CRP与单核细胞孵育48小时;
(3)不同浓度Ox-LDL与HDL共处理组:分别用500mg/L HDL+20mg/LOx-LDL、500mg/L HDL+40mg/L Ox-LDL、500mg/L HDL+80mg/L Ox-LDL与单核细胞孵育48小时;
(4)不同浓度CRP组与HDL共处理组:分别用500mg/L HDL+3mg/L CRP、500mg/L HDL+10mg/L CRP、500mg/L HDL+20mg/L CRP与单核细胞孵育48小时;
(5)提取细胞总RNA行RT-PCR检测,每个培养孔提取2次细胞RNA送检,即n=6。
2.3.5 HDL对Ox-LDL、CRP介导单核细胞MCP-1蛋白表达的干预作用
(1)不同浓度Ox-LDL组:终浓度分别为20mg/L、40mg/L、80mg/L的Ox-LDL与单核细胞孵育48小时;
(2)不同浓度CRP组:浓度分别为3mg/L、10mg/L、20mg/L的CRP与单核细胞孵育48小时;
(3)不同浓度Ox-LDL与HDL共处理组:分别用500mg/L HDL+20mg/LOx-LDL、500mg/L HDL+40mg/L Ox-LDL、500mg/L HDL+80mg/L Ox-LDL与单核细胞孵育48小时;
(4)不同浓度CRP组与HDL共处理组:分别用500mg/L HDL+3mg/L CRP、500mg/L HDL+10mg/L CRP、500mg/L HDL+20mg/L CRP与单核细胞孵育48小时:
(5)收集细胞上清进行ELISA检测,每个培养孔提取3次细胞上清送检,即n=9。
2.4.实时荧光定量RT-PCR法检测MCP-1和CCR2 mRNA表达
提取细胞样品总RNA,设计内参β-actin和目的基因引物,采用RT-PCR扩增,同时加入荧光染料,通过检测目的基因与内参基因达到荧光设定值所需循环数(Ct值)(ΔCt)并对不同处理组目的基因的ΔCt进行比较,可对未知标本靶基因的原始拷贝数做出判断。
2.5酶联免疫吸附试验(Enzyme-Linked ImmunosorbentAssays,ELISA)检测细胞培养液MCP-1蛋白含量,采用双抗体夹心ELISA法。
3.统计学方法
RT-PCR结果(ΔCt)和ELISA结果(ug/L)以均数±标准差表示,进行方差齐性检验。方差齐(P＞0.10)则采用方差分析,方差不齐则采用Tamhane法校正。若方差分析结果提示各组间差异具有显著性,则用Bonferroni法进行组间多重比较。采用单因素方差分析分别对不同浓度Ox-LDL和不同浓度CRP的作用进行统计学处理;采用两样本t检验对HDL对单核细胞MCP-1基因、蛋白和CCR2基因表达进行统计学分析;采用两因素方差分析对HDL的干预作用进行统计学处理。所有统计学处理均采用SPSS 13.0统计软件进行分析。P＜0.05表明有统计学差异。
结果
1.不同浓度Ox-LDL均可以促进单核细胞MCP-1 mRNA的表达(F=88.90,P=0.000);与对照组相比,20mg/L、40mg/L、80mg/L Ox-LDL处理组MCP-1基因表达上调了1.97倍、3.16倍和4.07倍;组间多重比较结果显示,20mg/L与40mg/L、40mg/L与80mg/L差异具有统计学意义(P=0.000,P=0.007),即Ox-LDL促进MCP-1基因表达的作用存在浓度效应。
2.不同浓度Ox-LDL均可促进单核细胞CCR2 mRNA的表达(F=129.25,P=0.000);与对照组相比,20mg/L、40mg/L、80mg/L Ox-LDL处理组MCP-1基因表达上调了2.12倍、2.54倍和3.02倍;组间多重比较结果显示,20mg/L与40mg/L、40mg/L与80mg/L差异具有统计学意义(P=0.008,P=0.009),Ox-LDL促进CCR2基因表达的作用存在浓度效应。
3.3mg/L CRP对MCP-1 mRNA表达的影响不具有统计学意义,10mg/L、20mg/L CRP则可以促进单核细胞MCP-1 mRNA的表达(F=122.07,P=0.000);与对照组相比,10mg/L、20mg/L CRP处理组MCP-1基因表达上调了3.09倍和4.04倍;10mg/L与20mg/L之间差异具有统计学意义(P=0.002),即CR2促进MCP-1基因表达的作用存在一定的浓度效应。
4.3mg/L CRP对CCR2 mRNA表达的影响也不具有统计学意义,而10mg/L、20mg/L CRP则可以促进单核细胞CCR2 mRNA的表达(F=177.35,P=0.000);与对照组相比,10mg/L、20mg/L CRP处理组MCP-1基因表达上调了2.96倍和3.59倍;10mg/L与20mg/L之间差异具有统计学意义(P=0.015),即CRP促进CCR2基因表达的作用存在一定的浓度效应。
5.500mg/L的HDL对单核细胞MCP-1(t=0.01,P=0.992)和CCR2基因(t=-0.623,P=0.547)表达的影响没有统计学意义;
6.500mg/L HDL可以抑制Ox-LDL促进MCP-1和CCR2 mRNA表达的作用(F=36.02,P=0.000);20mg/L Ox-LDL+500mg/L HDL组MCP-1和CCR2 mRNA表达量为20mg/L Ox-LDL的57.2%和49.5%;40mg/L Ox-LDL+500mg/L HDL组MCP-1和CCR2 mRNA表达量为40mg/L Ox-LDL的77.0%和86.7%;80mg/LOx-LDL+500mg/L HDL组MCP-1和CCR2 mRNA表达量为80mg/L Ox-LDL的71.9%和85.6%:
7.500mg/L HDL可以抑制CRP对单核细胞MCP-1和CCR2 mRNA表达的促进作用(F=20.4 1,P=0.000);10mg/L CRP+500mg/L HDL组MCP-1和CCR2 mRNA表达量为10mg/L CRP的73.9%和82.8%;20mg/L CRP+500mg/L HDL组MCP-1和CCR2 mRNA表达量为20mg/L CRP的69.8%和83.2%:
8.各浓度的Ox-LDL均可以促进单核细胞产生MCP-1(F=476.90,P=0.000);20mg/L与40mg/L、40mg/L与80mg/L差异具有统计学意义(P=0.000);
9.10mg/L、20mg/L CRP均可以促进单核细胞产生MCP.1(F=152.19.P=0.000);3mg/L CRP对单核细胞MCP-1蛋白含量的影响没有统计学意义(P=0.203);10mg/L与20mg/L CRP差异具有统计学意义(P=0.000);
10.500mg/L HDL对单核细胞MCP-1蛋白表达的影响没有统计学意义(t=-0.585,p=0.567);
11.500mg/L HDL可抑制Ox-LDL促进MCP-1蛋白表达的作用(F=14.12,P=0.000);
12.500mg/L HDL可抑制CRP促进MCP-1蛋白表达的作用(F=10.88,P=0.002)。
结论
1.在体外,Ox-LDL和C反应蛋白均可以促进单核细胞MCP-1和CCR2 mRNA的表达和MCP-1的分泌,对炎症反应起着重要的推动作用;
2.HDL对单核细胞MCP-1/CCR2没有直接作用,但可以抑制Ox-LDL、CRP上调MCP-1和CCR2基因表达的作用,这可能是HDL与AS发病呈负相关的机制之一;
3.CRP、Ox-LDL和HDL对MCP-1作用的信号转导通路需要进一步研究。
Background
As one of the most common cardiovascular diseases,Coronary heart disease (CHD) has been seriously jeopardizing health and quality of life of human beings. The pathogenesis of CHD,However,is not thoroughly identified.It is of much importance to study the relevant risk factors of CHD.Data from several studies,such as the Framingham Heart Study,have demonstrated some independent risk factors of CHD and predictors for cardiovascular outcomes,such as elevated blood pressure, diabetes mellitus,smoking,high low-density lipoprotein,reduced high-density lipoprotein,age and gender.Recently,some researchers believe that inflammation is involved in the pathogenesis and progress of CHD.So we have paid much attention on the inflammatory mechanism of CHD and try to illustrate pathogenesis of CHD in terms of relationships among traditional risk factors such as lipid,C-reactive protein (CRP) and inflammation.
Atherosclerosis(AS) is pathological basis of CHD.Since Ross raised the response-to-injury theory of AS in 1999,increasing evidence suggests AS is an inflammatory process after vascular injury.Endothelial injury and oxidative modification of low-density lipoprotein(LDL) are considered the earliest stage of AS pathogenesis.Low-density lipoprotein(LDL),which may be modified by oxidation, glycation(in diabetes),aggregation,association with proteoglycans,or incorporation into immune complexes,is a major cause of injury to the endothelium and underlying smooth muscle.When LDL particles become trapped in an artery,they can undergo progressive oxidation and be internalized by macrophages by means of the scavenger receptors on the surfaces of these cells.The internalization leads to the formation of lipid peroxides and facilitates the accumulation of cholesterol esters,resulting in the formation of foam cells.Oxidized-LDL(Ox-LDL) is chemotactic for other monocytes and can up-regulate the expression of genes for macrophage colony-stimulating factor and monocyte chemoattractant protein-1(MCP-1) derived from endothelial cells,and other inflammatory cytokines such as tumor necrosis factor-α(TNF-α), interleukin-6(IL-6),vascular cell adhesion molecule-1(VCAM-1) and intercellular cell adhesion molecule-1(ICAM-1).These cytokines may help expand the inflammatory response by stimulating the replication and secretion of monocyte-derived macrophages and the entry of new monocytes into lesions.Those activated macrophages have enhanced secretion property leading to increased level of MCP-1,ICAM-1 and VCAM-1.Such a positive-feedback results in the aggregation and adhesion to injury endothelium of monocyte-macrophages.These macrophages transformed into foam cells and,thus,led to the AS.So,serum level of MCP-1 could partly reflect the extent and amplitude of inflammation.The C-C chemokine receptor 2,commonly known as CCR2,is the specific receptor for MCP-1 and widely expression on endothelial cells,monocytes and smooth muscle cells.MCP-1/CCR2 plays an important role in pathogenesis and progress of inflammation relevant diseases,including AS.
C-reactive protein(CRP) is a sensitive marker for inflammation and tissue injury. A large number of research indicated that CRP was an independent risk factor for AS. The American heart Association(AHA) recommended the use of CRP as a prognostic marker in patients with acute coronary syndrome(ACS) in addition to other prognostic factors,including troponin levels.An evaluation of CRP levels at the time of admission should be included in the evaluation of the patient risk profile,including clinical data,associated diseases,markers of myocardial necrosis,left ventricle performance,and age.A cutoff level of 10mg/L was identified as marker of higher risk for death and possibly myocardial infarction in ACS,whereas a cutoff of 3 mg/L identifies a group of patients at intermediate risk and a high rate of recurrent events.
Other researches indicated that CRP enhanced gene expression of Lectin- like oxidized low density lipoprotein receptor—1(LOX—1) leading to intracellular accumulation of cholesterol,induced apoptosis of vascular smooth muscle cell and VCAM-1 gene expression through nuclear factor-κB pathway.CRP was also found to attenuate nitric oxide synthesis in endothelial cell and repair in injured endothelium. These findings provided direct evidence for the hypothesis that CRP may not only a marker and predictor,but a direct cause for AS through upregulate inflammatory response.However,little is known about the relationship between CRP and MCP-1.
The results of numerous epidemiological studies have established that high density lipoprotein(HDL) levels correlate inversely with the risk of developing cardiovascular disease.This relationship reflects several functions of HDL,the most extensively studied of which is their ability to remove excess cholesterol from cells, such as macrophages in the artery wall,in the first step of the reverse cholesterol transport pathway.In recent years it has become increasingly apparent that HDL also has potent anti-inflammatory properties.This has been demonstrated in vitro as well as in vivo.For example,the cytokine-induced expression of VCAM-1,ICAM-1,and E-selectin in cultured human umbilical vein endothelial cells(HUVECs) is inhibited by HDL in a concentration-dependent manner.It remains uncertain that whether HDL attenuates MCP-1 expression.
In summary,we hypothesized that CRP could enhance MCP-1 and CCR2 gene expression,which result in the aggregation and activation of monocytes in early stage of AS.HDL has potent anti-inflammatory properties through inhibiting MCP-1 and CCR2 expression.
Objectives:
1.To determine the effects of Ox-LDL and CRP on gene and protein expression of MCP-1 and CCR2 in monocytes.
2.To investigate whether HDL could regulate gene and protein expression of MCP-1 and CCR2 in monocytes which was elicited by Ox-LDL and CRP.
Methods:
1.Preparation of Ox-LDL
We separated and quantified the LDL from blood of healthy individuals by density gradient ultracentrifugation.Adjusted LDL to a final concentration of 500mg/L,then incubated it with 10mmol/L CuSO_4.The oxidation was ceased by adding 100μmol/L EDTA.Finally,Bradford method was utilized to detect the protein level of Ox-LDL.
2.Isolation and culture of monocytes
Monocytes were isolated from human peripheral blood by Ficoll-Hypaque density gradient centrifugation,inoculated in RPMI-1640 medium supplemented with 1%Penicillin and 10%fetal bovine serum(FBS) and incubated under the condition of 5%CO2,37.5℃.
3.Groups and cultural condition
(1)Monocytes were cultured with equal RPMI-1640 medium as control.
(2)Different concentration groups of Ox-LDL:Monocytes were treated with Ox-LDL (20mg/L、40mg/L and 80mg/L) for 48h,respectively.
(3)Different concentration groups of CRP:Monocytes were treated with CRP (3mg/L、10mg/L、20mg/L) for 48h,respectively.
(4)500mg/L HDL was added alone and cultured with monocyte to determine its role on both genes expression and MCP-1 protein level;
(5)Different concentration groups of Ox-LDL and CRP as described in(2) and(3), each group added 500mg/L HDL before culture.
4.Real-time quantitative PCR for MCP-1 and CCR2
MCP-1 and CCR2 mRNA was examined by Fluorescent quantitative RT-PCR. Total RNA extracted from cultured cells was reverse-transcripted and amplified using specific primers for human MCP-1 and CCR2.The 2(-Delta Delta C(T)) method is utilized to analyze the relative changes in gene expression from real-time quantitative PCR experiments.
5.ELISA assay for MCP-1 protein level
This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for MCP-1 has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any MCP-1 present is bound by the immobilized antibody.An enzyme-linked monoclonal antibody specific for MCP-1 is added to the wells.Following a wash to remove any unbound antibody-enzyme reagent,a substrate solution is added to the wells and color develops in proportion to the amount of MCP-1 bound in the initial step.The color development is stopped and the intensity of the color is measured.
6.Data analysis
SPSS 13.0 statistical software was used to analyze data.Data are presented as Mean±S.D.Statistical significance was determined in a multiple comparisons among different groups of data in which one-way ANOVA test indicated the presence of significant differences.P value＜0.05 was considered to be significant.
Results:
1.Effects of Ox-LDL on MCP-1 and CCR2
Incubation of monocytes with Ox-LDL of different concentration for 48h significantly enhanced the expression of MCP-1(mRNA and protein) and CCR2 mRNA in a concentration-dependent manner(P=0.000).When compared with control,MCP-1 mRNA expression increased to 1.97,3.16 and 4.07 fold and protein expression increased(P=0.000) while CCR2 mRNA expression increased to 2.12, 2.54 and 3.02 fold(P=0.009) after treated with Ox-LDL of different concentrations.
2.Effects of CRP on MCP-1 and CCR2
Incubation of monocytes with CRP of different concentration for 48h significantly enhanced the expression of MCP-1(mRNA and protein) and CCR2 mRNA in a concentration-dependent manner(P=0.000).There was no significance between 3mg/L CRP group and control MCP-1 mRNA expression increased to 3.09 and 4.04 fold(P=0.000) and protein expression elevated(P=0.000) while CCR2 mRNA expression increased to 2.96 and 3.59 fold(P=0.005) after treated with CRP of different concentrations(10mg/L and 20mg/L),as compared with control.
3.HDL had no direct influence on on both genes expression and MCP-1 protein level;
4.Role of HDL in expression of MCP-1 and CCR2 elicited by Ox-LDL and CRP
Co-incubation of monocytes with HDL significantly inhibited Ox-LDL-induced and CRP-induced expression of MCP-1(mRNA and protein) and CCR2 mRNA.
Conclusions:
1.Oxidized low-density lipoprotein upregulates the mRNA and protein expression of MCP-1 and mRNA expression of CCR2;
2.C-reactive protein upregulates the mRNA and protein expression of MCP-1 and mRNA expression of CCR2;
3.High density lipoprotein which inhibits Ox-LDL-induced and CRP-induced upregulation of MCP-1 and CCR2 may exert beneficial effect in the prevention and treatment of atherosclerosis.
引文
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[2]Ross R.Atherosclerosis～an inflammatory disease[J].N Engl J Med, 1999,340(2): 115-26.
[3]Coll B, Alonso-Villaverde C, Joven J.Monocyte chemoattractant protein-1 and atherosclerosis: is there room for an additional biomarker?[J].Clin Chim Acta,2007, 383(1-2): 21-9.
[4]Mehrabian M, Sparkes RS, Mohandas T, et al.Localization of monocyte chemotactic protein-1 gene (SCYA2) to human chromosome 17q 11.2-q21.1 [J].Genomics, 1991, 9(1): 200-3.
[5]Hoogeveen RC, Morrison A, Boerwinkle E, et al.Plasma MCP-1 level and risk for peripheral arterial disease and incident coronary heart disease:Atherosclerosis Risk in Communities study[J].Atherosclerosis, 2005, 183(2):301-7.
[6]Arakelyan A, Petrkova J, Hermanova Z, et al.Serum levels of the MCP-1 chemokine in patients with ischemic stroke and myocardial infarction [J].Mediators Inflamm, 2005, 2005(3): 175-9.
[7]Fleming I, Mohamed A, Galle J, et al.Oxidized low-density lipoprotein increases superoxide production by endothelial nitric oxide synthase by inhibiting PKCalpha[J].Cardiovasc Res, 2005, 65(4): 897-906.
[8]Apostolakis S, Papadakis EG, Krambovitis E, et al.Chemokines in vascular pathology (review)[J].Int J Mol Med, 2006, 17(5): 691-701.
[9]Mackness B, Hine D, Liu Y, et al.Paraoxonase-1 inhibits oxidised LDL-induced MCP-1 production by endothelial cells[J].Biochem Biophys Res Commun,2004,318(3):680-3.
[10]Kusano KF,Nakamura K,Kusano H,et al.Significance of the level of monocyte chemoattractant protein-1 in human atherosclerosis[J].Circ J,2004,68(7):671-6.
[11]朱惠莲,唐志红,夏敏,等.LOX-介导ox-LDL诱导的血管内皮细胞MCP-的表达[J].中国病理生理杂志,2005(07).
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