【作者】 何军；

【导师】 马业新；

【作者基本信息】 华中科技大学 ， 内科学， 2007， 博士

【摘要】 研究背景和目的动脉粥样硬化(Atherosclerosis, As)是心血管疾病的早期病理过程,从As产生到出现临床症状要经过数十年的发展,在此漫长的过程中,人们往往忽略危险的存在,又因传统的防治措施作用有限,使As的发展难以遏制。减缓乃至逆转As的发生发展是防治心血管疾病,提高人们生活质量的客观要求,因此,明确As发生发展机制,寻找早期有效的防治As的靶点十分必要。通过从基因水平、细胞水平和整体水平对As广泛而深入的研究发现,内皮细胞、巨噬细胞和平滑肌细胞(Smooth musle cells, SMCs)是构成粥样斑块的主要细胞成分,血管内皮功能失调、SMCs增殖和脂质沉积是粥样斑块形成的三个重要环节,巨噬细胞转化为泡沫细胞是其中的关键步骤。但是,这一系列过程的详细机制仍不十分清楚。目前有关As的研究较多集中在血管内皮细胞损伤和SMCs增殖,对巨噬细胞转化为泡沫细胞的机制及其调控研究较少,国内少见报道。近年研究发现,非对称性二甲基精氨酸(Asymmetric dimethylarginine, ADMA)血浆水平在冠心病、高血压、高血脂、糖尿病、高同型半胱氨酸血症、胰岛素抵抗、心力衰竭、肾功能衰竭时明显升高,其作用及机制成为人们关注的焦点。ADMA是内源性一氧化氮合酶(Nitric oxide synthase, NOS)的竞争性抑制剂,可致内皮功能受损、SMCs增生,在巨噬细胞泡沫化中的作用尚不清楚。NOS分为神经型(Neural nitric oxide synthase, nNOS)、内皮型(Endothelial oxide synthase, eNOS)和诱导型(Inducible oxidesynthase, iNOS)三种。nNOS和eNOS是机体正常存在的,大多数学者认为iNOS在正常情况下几乎不表达,当细胞受到炎症因子、免疫因子等刺激时表达增加。氧化型低密度脂蛋白(Oxidized low density lipoprotein, oxLDL)对粥样硬化形成过程中泡沫细胞聚集有重要作用,细胞摄取oxLDL由数种清道夫受体介导,血凝素样低密度脂蛋白受体-1 (Lectin-like low density lipoprotein recepter-1, LOX-1)是其中最特异的oxLDL受体。LOX-1主要在人冠状动脉内皮细胞、牛主动脉内皮细胞、兔和大鼠主动脉表达,在巨噬细胞、血小板和SMCs也有少量表达。研究表明,ADMA可上调血管内皮细胞、SMCs和巨噬细胞株NR8383 LOX-1表达,提示ADMA与As可能密切相关。本研究拟采用分子生物学实验方法,用ADMA干预oxLDL预处理的原代培养大鼠腹腔巨噬细胞,观察巨噬细胞iNOS和LOX-1表达,以探讨ADMA对巨噬细胞泡沫化的影响及机制;给高脂饲养的大鼠ADMA后,检测大鼠主动脉脂质沉积及iNOS和LOX-1表达,以期对ADMA在大鼠粥样硬化中的作用及机制进行初步探讨,为ADMA成为心血管疾病防治靶点提供可能的理论依据。方法1.体外细胞学实验:Wistar大鼠42只。分离培养大鼠腹腔巨噬细胞。实验分组及处理:①对照组:巨噬细胞与PBS (PH6.8)孵育48h。②oxLDL组:巨噬细胞与oxLDL(终浓度50μg/mL)共育48h。③0+A组:先以oxLDL(终浓度50μg/mL)与巨噬细胞共育24h,再加入ADMA(终浓度15μmol/L)继续孵育24h。④0+A+L组:先以oxLDL(终浓度50μg/mL)与巨噬细胞共育24h,再加入ADMA(终浓度15μmol/L)和L-Arg(终浓度1.2mmol/L)继续孵育24h。收集细胞和培养液,测定细胞内胆固醇含量及培养液NO水平和iNOS活性,提取细胞总RNA和总蛋白,分别通过半定量RT-PCR和Western-blot检测巨噬细胞iNOS,LOX-1 mRNA和蛋白表达,以β-actin为内参照进行标化。每组重复3次。2.动物体内实验:Wistar大鼠48只,随机分为四组:①正常对照组(n=8):标准大鼠饲料,正常饮水。②高脂组(n=12):高脂饲料,正常饮水。③ADMA组(n=14):高脂饲料,正常饮水,ADMA (0.2mg/Kg/d)灌胃,每日一次。④ADMA+L-Arg组(n=14):高脂饲料,饮水中含3% L-Arg,ADMA(0.2mg/Kg/d)灌胃,每日一次。饲养满18周后取血,测定大鼠血清总胆固醇、甘油三酯、NO及iNOS水平,取胸主动脉做病理切片,观察其病理变化。以半定量RT-PCR和Western-blot分别检测主动脉iNOS和LOX-1 mRNA及蛋白表达,以β-actin为内参照进行标化。结果1.体外细胞学实验:①oxLDL组和O+A组巨噬细胞内胆固醇含量较正常对照组明显增加(均p<0.01),且O+A组较oxLDL组升高更明显(p<0.05),而O+A+L组胆固醇含量较O+A组降低(p<0.05)。②与正常对照组和oxLDL组相比,0+A组NO含量降低(p<0.01或p<0.05);0+A+L组NO含量较0+A组显著升高(p<0.05)。③与正常对照组和oxLDL组相比,0+A组iNOS活力明显升高(均p<0.05),0+A+L组iNOS活力较0+A组降低(p<0.05)。双变量相关性分析显示,NO含量与iNOS活力呈负相关(r=-0.697, p=0.012)。④与正常对照组和oxLDL组相比, O+A组iNOS mRNA和蛋白表达量显著增加,其差异均有显著性(均p<0.01);O+A+L组iNOS mRNA和蛋白表达较O+A组降低(均P<0.01)。⑤O+A组LOX-1 mRNA和蛋白表达较正常对照组和oxLDL组增强,其差异均有显著性(均p<0.05);与O+A组相比,O+A+L组LOX-1 mRNA和蛋白表达降低,两组差异有显著性(p<0.001或p<0.05)。2.动物体内实验:①主动脉病理切片显示正常对照组血管内膜光滑、完整,无脂质沉积;高脂组血管内膜光滑、完整,有少量脂质沉积;ADMA组血管内膜断裂,内膜下有明显脂质沉积,血管中层SMCs增生明显,且排列紊乱;ADMA+L-Arg组血管内膜完整,脂质沉积和SMCs增生较ADMA组减弱。②ADMA组大鼠血清NO水平和iNOS活性明显升高,与正常对照组和高脂组比较有统计学差异(p<0.01或p<0.05),ADMA+L-Arg组NO水平和iNOS活性较ADMA组明显下降(均p<0.05)。双变量相关性分析显示,NO与iNOS间呈正相关(r=0.512, p=0.003)。③高脂组、ADMA组和ADMA+L-Arg组血清TG和TC水平均升高,与正常对照组相比有显著性差异(均p<0.01)。④ADMA组大鼠主动脉iNOS mRNA和蛋白表达量与正常对照组和高脂组相比显著上调(p<0.001或p<0.05);ADMA+L-Arg组iNOS mRNA和蛋白表达量较ADMA组明显降低(均p<0.01)。⑤ADMA组大鼠主动脉LOX-1 mRNA和蛋白表达量较正常对照组和高脂组显著上调(p<0.01或p<0.001);与ADMA组相比,ADMA+L-Arg组LOX-1 mRNA和蛋白表达量明显降低,其差异有显著性(p<0.01或p<0.05)。结论①ADMA可促进培养的大鼠腹腔巨噬细胞摄取oxLDL,促进巨噬细胞泡沫化。②ADMA可促进高脂饲养大鼠主动脉脂质沉积。③上调iNOS和LOX-1表达可能是ADMA促进泡沫细胞形成和脂质沉积的内在机制之一,有效抑制ADMA的作用可能是早期防治As的靶点。更多还原

【Abstract】 Background & ObjectiveAtherosclerosis (As) is an early pathological stage of cardiovascular disease, it always undergoes several decades years from early atherogenesis to clinical case. During this long time, people usually ignore the existence of dangers, and the conventional preventives are always not so effective as people expected. In order to promote people’s life quanlity, slowing down and reversing the genesis and development of As is an inevitable trend. So, it is necessary and important to understand the mechanism of As, and to get a target of prevention and therapy. On the basis of wide and deep studies on genes, cells and systems, we know that endothelial cells, macrophages and vascular smooth muscle cells (VSMCs) are the major components of fatty streak. Endothelial dysfunction, VSMCs proliferation and lipidosis are the three important steps of lesion formation, the key step is macrophages transforming to foam cells with mechanism not well known yet. Nowadays, researches about As are much more focused on endothelial dysfunction and VSMCs proliferation, and less on the mechanism and regulation of macrophages transforming to foam cells. Recent researches demonstrated that the plasma levels of asymmetric dimethylarginine (ADMA) are increased in cardiovascular diseases, such as coronary heart disease, hypertension, hypercholesterolemia, diabetes mellitus, insulin resistance, hyperhomocysteinemia, heart failure and renal failure. The role and mechanism of ADMA are becoming a focus. ADMA is a competitive inhibitor of endogenous nitric oxidize synthase (NOS), it can lead to endothelial dysfunction and VSMCs proliferation, while it’s effect on macrophages transforming to foam cells is unknown. NOS includes neural NOS (nNOS), inducible NOS (iNOS) and endothelial NOS (eNOS). Different from other two types, iNOS has little expression during physiological state, but increases significantly when cells are stimulated by inflammatory factors and immune factors. Oxidized low-density lipoprotein (oxLDL) is believed to play a key role in accumulation of foam cells in atherogenesis. The cellular uptake of oxLDL is mediated by so-called scavenger receptors. Among them, Lectin-like low density lipoprotein receptor-1 (LOX-1) is the most specific receptor of oxLDL. LOX-1 is present in the endothelium of human coronary arteries, rabbit and rat aorta and bovine endothelial cells. A small amount of LOX-1 has also been identified in macrophages, platelets and VSMCs. Studies showed that ADMA upregulates LOX-1 expression in endothelial cells, VSMCs and macrophages, which suggested that ADMA is related to As closely. In the present study, we used methods of molecular biology to interfere cultured rat celiac macrophages pretreated by oxLDL with ADMA, and then observed the expressions of iNOS and LOX-1, so as to discuss the mechanism of macrophages transforming to foam cells. By measuring rat aorta lipidosis and expression of iNOS and LOX-1 after being fed by ADMA , to probe into the effect and mechanism of ADMA in atherogenesis. We hope to provide a reliable clue for regarding ADMA as a target of cardiovascular disease’s prevention and therapy.Methods1. Study in vitroForty-two Wistar rats were enrolled. Celiac macrophages of rats were gathered and cultured. Groups and interference:①Control group: Macrophages were incubated with PBS solution for 48 hours.②oxLDL group: Macrophages were treated by oxLDL (final concentration 50μg/mL) for 48 hours.③ADMA group: Macrophages were pretreated by oxLDL (final concentration 50μg/mL) for 24 hours, then co-incubated with ADMA (final concentration 15μmol/L) for 24 hours.④0+A+L group: Macrophages were pretreated by oxLDL (final concentration 50μg/mL) for 24 hours, then co-incubated with ADMA (final concentration 15μmol/L) and L-Arg (final concentration 1.2mmol/L) for 24 hours. Each group was repeated for 3 times. Immunocytochemistry staining of macrophages were done. Intracellular cholesterol level, NO level and iNOS activity in medium were measured. Total RNA and protein of macrophages were extracted, expression of iNOS, LOX-1 mRNA and protein were analyzed by semi-quantified RT-PCR and Western-blot respectively, ratβ-actin were used as internal standard.2. Study in vivoForty-eight Wistar rats were enrolled and divided into four groups randomly.①Control group (n=8): Rats were fed by normal feedstuff and water.②High fat diet group (n=12): Rats were fed by high fat feedstuff and normal water.③ADMA group (n=14): Rats were fed by high fat feedstuff and normal water, ADMA (0.2mg/Kg/d) was infused into the stomachs once a day.④ADMA +L-Arg group (n=14): Rats were fed by high fat feedstuff, water with 3% L-Arg , ADMA (0.2mg/Kg/d) was infused into the stomachs once a day. Eighteen weeks later, serum levels of cholesterol, triglyceride, NO and iNOS were examined respectively, slices of aorta were inspected. Total RNA and protein of aorta were extracted, expression of iNOS, LOX-1 mRNA and protein were analyzed by semi- quantified RT-PCR and Western-blot respectively, normalized by the internal control geneβ-actin.Results1. Study in vitro①Intracellular cholesterol level in oxLDL group and ADMA group were higher than that in control group (p<0.01, either), it increased significantly in ADMA group compared with oxLDL group (p<0.05), but in O+A+L group it was lower than that in ADMA group (p<0.05).②NO level in ADMA group dropped down markedly compared with control group and oxLDL group (p<0.01 or p<0.05), it was much higher in O+A+L group than that in ADMA group (p<0.05).③INOS activity increased in ADMA group compared with control group and oxLDL group (p<0.05, either), but decreased significantly in O+A+L group compared with ADMA group (p<0.05). Bivariate correlation analysis showed that NO and iNOS was negative related (r=-0.697, p=0.012).④Expression of iNOS mRNA and protein increased in ADMA group compared with control group and oxLDL group (p<0.01, either), but decreased in O+A+L group compared with ADMA group (p<0.01, either).⑤Expression of LOX-1 mRNA and protein increased in ADMA group compared with control group and oxLDL group (p<0.05, either), but decreased in O+A+L group compared with ADMA group (p<0.001 or p<0.05).2. Study in vivo①Aorta slices were stained by sudauⅢ. Inspected by optic microscope, the intima in control group was smooth and integrated, without lipid sediment. There were small amount of lipidosis in high fat diet group. In ADMA group, the intima was broken, with obvious lipidosis, VSMCs proliferation of media was marked and the array was irregular. In ADMA +L-Arg group, the intima was intact, lipidosis and VSMCs proliferation were lower than that in ADMA group.②Serum levels of NO and iNOS in ADMA group elevated compared with high fat diet group and control group (p<0.01or p<0.05), but the levels dropped down in ADMA +L-Arg group compared with ADMA group (p<0.05). Bivariate correlation analysis showed that NO and iNOS was positive related (r=0.512, p=0.003).③Serum levels of TG and TC in high fat diet group, ADMA group and ADMA +L-Arg group were higher than those in control group (p<0.01, either).④INOS mRNA and protein expression of aorta enhanced notably in ADMA group compared with control group and high fat diet group (p<0.05 or p<0.001), but they reduced in ADMA +L-Arg group compared with ADMA group (p<0.01, either).⑤LOX-1 mRNA and protein expression of aorta increased in ADMA group compared with high fat diet group and control group (p<0.01 or p<0.001), but dropped dramatically in ADMA +L-Arg group compared with ADMA group (p<0.01 or p<0.05). Conclusions①ADMA promotes celiac macrophages of rats to intake oxLDL and induce foam cell formation.②ADMA accelerates atherogenesis in high fat diet rat.③Upregulating iNOS and LOX-1 expression might be one of the underlying mechanisms of the process. Supressing the action of ADMA may be an effective target of As prevention.更多还原