【作者】 靳欣；

【导师】 刘运海；

【作者基本信息】 中南大学 ， 神经病学， 2012， 博士

【摘要】 背景：脑卒中是严重危害我国国民生命与健康的主要疾病,已成为我国国民的首位死因。脑梗死占全部脑卒中的50-70%,以动脉粥样硬化病变为病理基础的脑梗死是最常见的类型。因此,深入研究动脉粥样硬化的发病机制是脑梗死防治的一项重大课题。脂肪组织不仅是储存能量的器官,同时还以旁、自分泌和远距离分泌方式分泌生物活性因子—脂肪细胞因子。多种脂肪细胞因子如瘦素、脂联素和肿瘤坏死因子等能通过影响机体的脂质代谢,胰岛素抵抗,炎症反应等参与代谢综合征、动脉粥样硬化及相关疾病的发生与发展,脂肪细胞因子的研究已成为动脉粥样硬化性疾病的研究热点。Chemerin是一种新发现的脂肪细胞因子,其功能受体为G蛋白受体家族成员Chemerin样受体(CMKLR1/ChemR23), Chemerin通过作用于表达ChemR23的细胞参与机体的免疫反应及能量代谢等活动。目前研究表明,在肥胖、糖尿病及冠心病患者体内Chemerin水平有显著升高；冠状动脉的斑块上其表达丰富,提示Chemerin可能和动脉粥样硬化病变发展有关。我们推测Chemerin与颈动脉粥样硬化病变及脑梗死的发生与发展相关,关于Chemerin与脑梗死和脑梗死患者的动脉粥样硬化病变关系的研究目前国内外均无相关报道。内皮细胞损伤及随后的持续性炎症反应被认为是动脉粥样硬化的始动因素,多种脂肪细胞因子被证实可直接影响或协同促进血管内皮的功能紊乱诱发动脉粥样硬化进程。Chemerin是机体重要的免疫因子,在多种自身免疫疾病及炎性疾病的早期阶段介导固有免疫及适应免疫,招募未成熟免疫细胞,促进免疫细胞成熟等方面广泛参与机体的免疫反应。目前研究提示Chemerin可能在动脉粥样硬化病变中具有重要作用,但Chemerin对血管内皮细胞功能是否具有影响这一关键环节尚无研究。本研究是国内外首次对Chemerin与脑梗死和脑梗死患者的动脉粥样硬化病变关系探讨,以期为脑梗死及动脉粥样硬化的研究提供新的靶点,并且在国内外首次于细胞水平上探讨Chemerin对基础情况下及炎性损伤情况下的血管内皮细胞的影响,从而开拓方向,为下一步的研究提供依据。第一部分血浆Chemerin水平与脑梗死及颈动脉粥样硬化关系的研究目的：检测脑梗死患者血浆Chemerin水平,探讨其与脑梗死及颈动脉粥样硬化的关系,分析Chemerin与代谢指标及炎性指标的相关性,为阐明Chemerin对脑梗死和动脉粥样硬化的影响提供依据。方法：选择148例脑梗死急性期患者为病例组和57例健康对照组,采取酶联免疫吸附试验(ELISA)检测血浆Chemerin水平,分析与脑梗死的关系,通过相关性分析,研究Chemerin与代谢及炎性指标之间的关系。脑梗死组采用多普勒超声检测颈动脉,计算双侧颈总动脉平均内-中膜厚度(IMT)并依据颈动脉是否存在斑块及其性质分亚组(无斑块组46例,稳定斑块组39例和不稳定斑块组63例),通过Logistic回归分析Chemerin水平与脑梗死及斑块性质之间的关系。结果：(1)与正常对照组比较,脑梗死患者血浆Chemerin水平明显增高(P<0.01), Logistic回归分析在考虑了高血压史、糖尿病史、冠心病史、高脂血症、吸烟史、血浆超敏C反应蛋白(hsCRP)水平后,Chemerin水平增高并不能使脑梗死的发病风险增加。(OR:1.183;95%CI:0.743-1.885; P=0.479)(2)血浆Chemerin水平与低密度脂蛋白胆固醇(LDL-C),收缩压及炎性因子hsCRP和肿瘤坏死因子-α (TNF-α)水平正相关(P<0.05),与体重指数,空腹血糖,总甘油三酯,胆固醇,高密度脂蛋白胆固醇,舒张压水平无关；(3)脑梗死患者的血浆Chemerin水平与颈总动脉平均IMT呈正相关(r=0.187；P=0.023)；(4)脑梗死患者中不稳定斑块组的Chemerin水平高于稳定斑块组及无斑块组患者,而稳定斑块组与无斑块组之间无明显差别,经Logistic回归分析在考虑了hsCRP、低密度脂蛋白胆固醇水平后,Chemerin水平并不能使不稳定斑块的风险增加(OR：1.018；95%CI：0.920-1.127：P=0.729)。结论：(1)脑梗死患者和颈动脉具有不稳定斑块的脑梗死患者拥有较高的血浆Chemerin水平；(2)脑梗死患者的血浆Chemerin水平与代谢指标和炎性指标相关；(3)脑梗死患者的血浆Chemerin水平可能可以预测动脉粥样硬化的严重程度。第二部分Chemerin对HUVEC功能影响的初步探讨目的：通过测定与分析多种内皮细胞分泌因子的水平,包括：细胞间粘附分子-1(intercellular adhesion molecule-1,ICAM-1)和血管细胞粘附分子-1(vascular cell adhesion molecule-1,VCAM-1),一氧化氮(nitric oxide,NO),炎性因子白介素-6(Interleukin-6, IL-6)探索不同浓度Chemerin干预对基础水平的人脐静脉内皮细胞功能的影响及生理浓度的Chemerin对肿瘤坏死因子-α (tumor necrosis factor-alpha, TNF-α)诱导的血管内皮细胞损伤模型的影响。通过检测并分析细胞内生存关键通路磷脂酰肌醇3激酶途径(Phosphatidylinositol3-kinase/Akt,P13K/Akt)中AKT,磷酸化AKT (p-AKT),内皮型一氧化氮合酶(Endothelial nitric oxide synthase,eNOS)及磷酸化内皮型一氧化氮合酶(p-eNOS)表达水平,探讨Chemerin的可能作用机制。方法：(1)培养人脐静脉内皮细胞-12细胞株(Human Umbilical Vein Endothelial Cells-12, HUVEC-12),依据不同浓度的重组Chemerin分别干预24小时或48小时,共12组,Chemerin浓度(0,5,25,50,100,200ng/ml)分别为24小时①-⑥组,48小时⑦-12组,应用Griess法测定细胞培养液上清液中NO水平；ELISA试剂盒检测细胞内IL-6、 ICAM-1、VCAM-1水平。通过单因素方差分析及多重比较,得出Chemerin对基础水平的血管内皮细胞的影响,并结合既往资料与实验结果推算最适干预浓度与时间(浓度：Chemerin100ng/ml,干预时间：24小时)；(2) Chemerin对TNF-a诱导的HUVEC-12细胞损伤模型的影响：设A为未加药物的空白对照组；B为Chemerin组：给予终浓度为(100ng/ml) Chemerin干预；C为TNF-a组给予终浓度为(50ng/ml)TNF-a干预；D为Chemerin+TNF-a组给予终浓度分别为100ng/mlChemerin+50ng/mlTNF-a干预；E为LY294002组,LY294002为P13K特异性抑制剂,给予终浓度分别为100ng/mlChemerin+50ng/ml TNF-a+20μmol/LLY294002干预。于24小时应用上述方法检测细胞培养上清液中NO水平；HUVEC的IL-6、ICAM-1、VCAM-1水平,应用Real-time PCR检测各组HUVEC-12的AKTmRNA表达水平,Western Blot法测定AKT,p-AKT,eNOS,p-eNOS蛋白水平。通过统计学软件计算分析结果,以P<0.05为有统计学意义。结果：(1) Chemerin能上调HUVCE-12细胞培养上清液中的NO水平,并且在5ng/ml24小时即明显增高,其诱导作用在(0-100ng/ml)范围内呈浓度依赖(P<0.05),在25-200ng/ml浓度范围48小时组较24小时组增高无统计学意义(P<0.05),而5ng/ml组48小时组较24小时组NO的水平增高(P<0.05),各组IL-6、ICAM-1、VCAM-1之间相比无差异(P>0.05)；(2) Chemerin组较空白对照组,NO的水平明显上升(P<0.01),p-AKT、eNOS及p-eNOS的水平增加(P<0.01),对AKT蛋白及AKT mRNA的表达无影响(P>0.05)；(3) Chemerin+TNF-a组较TNF-a组IL-6、ICAM-1、VCAM-1、NO的水平均明显下降(P<0.01),p-AKT、eNOS及p-eNOS的水平增加(P<0.01),对AKT蛋白水平及AKTmRNA表达无影响(P>0.05)(4)LY294002组较Chemerin+TNF-a组ICAM-1, VCAM-1, NO增高(P<0.05),IL-6未见明显改变(P>0.05),下调p-AKT (P=0.000), p-eNOS及eNOS蛋白表达(P<0.05),但对AKT蛋白水平及AKTmRNA表达无影响(P>0.05)结论：(1) Chemerin能抑制炎性损伤的血管内皮细胞分泌粘附分子和炎性分子,可能对损伤的内皮细胞具有保护作用；(2) Chemerin可以上调NO的释放,以上作用与eNOS及p-eNOS活化有关,P13K/AKT-eNOS途径参与了上述作用；(3) Chemerin对炎性损伤下的内皮细胞具有保护潜能,可成为动脉粥样硬化研究的靶点。更多还原

【Abstract】 BackgroundStroke is the most fatal disease with an annual estimate of more than2million new cases and at least1.5million deaths in China from the the latest report at2008. In particular, about50%-70%of patients with stro-ke are ischemic. Atherosclerosis is the pivotal process in the development of ischemic stroke.Adipose tissue is not only a store for energy but also appears to be an active endocrine organ that produces many cytokines, which are kno-wn as adipokines. These adipokines appear to have systemic effects on the lymphoid organs, liver, muscles, gonads, brain, and vasculature. Adipokines have been implicated in the pathogenesis of atherosclerosis. It’s known that adipose tissue can cause vascular complications either directly by causing atherosclerosis and inducing inflammatory responses, or indirectly by promoting the development of insulin resistance. These atherosclerosis-inducing pathways are mediated by kinds of adipokines, including adiponectin, leptin, tumor necrosis-alpha and resistin.Chemerin is recently discovered as an adipokine. It regulates the immune system and participates in inflammation by promoting the recur-itment of tissue macrophages and plasmacytoid dendritic cells as well, it is also associated with adipogenesis. As studies have shown that the lev- els of Chemerin are higher in the obesity and the patients with coronary disease. Therefore, we hypothesized that an increase in Chemerin might reflect carotid atherosclerosis and Chemerin levels might be related to the incidence of cerebral infarction. Endothelial cells dysfunction is the key point of the development of atherosclerosis. It has been reported that kinds of adipokines have important roles as vasoactive hormones which may impair endothelial function directly by altering vasoactive factor production and inciting endothelial cell activation. Whether Chemerin which was originally identified as a chemoattractant impact endothelial fuction directly? PART01Relationship between Chemerin Levels and Cerebral Infarction and AtherosclerosisObjectives1、To examined the levels of Chemerin in patients with cerebral infarction.2、To investigated the correlation with specific cardiometabolic parameters and also examed the relationship between the levels of Chemerin and cartiod atherosclerosis.MethodsIn the present study,148patients, all of whom had acute cerebral infarction within3days participated in this study and enrolled57healthy controls with no cerebrovascular disease. Their serum Chemerin levels and clinical parameters were measured. The serum chemerin levels of two groups of individals were determined by ELISA. All the patients under-gone ultrasound in order to calculate the carotid intima-media thickness (IMT) and detect the characters and quantity of cartiod atherosclerotic plaques. To divide into three group as the standard whether with the ex-istence of vulnerable plaque, stable plaque and normal. The serum che-merin levels were assessed by logistic regression in order to elucidate the relationship between Chemerin and cerebral infartion as well as vu-lnera-ble plaques. ResultsSerum Chemerin levels correlated positively with the carotid IMT and systolic blood pressure, Low density lipoprotein cholesterol, high sensitive C-reactive protein and Tumor necrosis factor-α levels. The case group has higher chemerin levels than the control and the levels are hig-her in the vulnerable plaques group. Multiple regression showed Che-merin was not an independent risk factor of cerebral infartion (OR:1.183;95%CI:0.743-1.885; P=0.479) nor an independent risk factor of vulnerable plaques (OR:1.018;95%CI:0.920-1.127; P=0.729)Conclusions:Serum Chemerin levels have a significant correlation with several pro-inflammation markers and metabolic risk factors as well as the caro-tid IMT in patients with acute cerebral infarction. However, multiple binary logistic regression showed Chemerin was not an independent risk factor of cerebral infarction nor vulnerable plaques. Additional investiga-tions are necessary to fully elucidate the role of Chemerin in cerebrovas-cular disease. PART02The Effects of Chemerin on Human Umbilical Vein Endothelial CellsObjectives1、To investigate the effect of Chemerin on Human Umbilical Vein Endothelial cells, we measure the level of nitric oxide(NO), inter-leukin-6(IL-6), intercellular adhesion molecule-1(ICAM-1) and vascular cell adhesion molecule-1(VCAM-1) after the stimulation of chemerin at different concentration at24hours and48hours respectively in vitro. And then to decide the optimal action time course and concentration.2. To explore the effects of Chemerin on TNF-alpha-treated HU-VECs-12which is the model of the research on impaired endothelial function. To investigate the effects of P13K/AKT-eNOS signal trans-duction pathway on vascular endothelial cells pretreated by chemerin.Methods1、HUVEC cells were cultured in serum-free medium before treatments for24h or48h with control groups and five different concen-trations groups(5,25,50,100,200ng/ml) of Chemerin. The total NO con-tent in cell medium by methods of Griess reagent and the protein levels of intercellular adhesion molecule-1(ICAM-1)/vascular cell molecule-1(VCAM-1) and Interleukin-6(IL-6) of HUVECs were detected by ELISA. 2、HUVECs stimulated by TNF-a(50ng/ml) in vitro.The total NO content in cell medium and the protein levels of ICAM-1/VCAM-1/IL-6of HUVECs were detected in the presence and absence of cheme-rin(100ng/ml), phosphatidylinositol3’-kinase(P13K) inhibitor LY-294002(20μmol/l) by the methods which had been mentioned above Intracellular signal molecules of P13k/AKT-eNOS signal such as Akt, phosphorylated-Akt,eNOS and phosphorylated-eNOS protein expres-sions were detected by western blot. The mRNA expression of Akt were measured by methods of realtime quantitative chain reaction polymerase.Results:1、Chemerin of (0-200ng/ml) significantly upregulated the levels of NO content.The increased expression of NO protein in chemerin-induced HUVECs was in a dose-dependent manner (P<0.05), These effects could partially diminished by cotreatment with LY294002.The protein levels of IL-6,intercellular adhesion molecule-1(ICAM-1) and vascular cell molecule(VCAM-1) of HUVECs hasn’t been impacted.2、Chemerin(100ng/ml) significantly inhibited Tumor necrosis-alpha induced The expression of ICAM-1.VCAM-1and IL-6production (P<0.01). Chemerin also activated Akt and enhance the expression of phosphorylated-Akt,eNOS and phosphorylated eNOS protein (P<0.01) The expression of Akt protein and mRNA expression of Akt remain the same (P>0.05). These effects on the activate of P13k/Akt pathway signal could be partially diminished by cotreatment with LY294002,But the down-regulation of IL-6was not influenced.Conclusions:Chemerin may enhances the eNOS ability and attenuate the endothelin system and the overexpression of adhesion molecules in dysfunctional endothelial cells. Chemerin has the potential on improving endothelial function. P13K/Akt-eNOS signal transduction pathway may participate in the activity.A detailed molecular and functional analysisi of the role of chemerin in endothelial function should provide futher understanding of the relationship between Chemerin and endothelial function.更多还原