【作者】 刘俊；

【导师】 李建军；

【作者基本信息】 北京协和医学院 ， 内科学， 2013， 博士

【摘要】 背景：前蛋白转化酶枯草溶菌素9(proprotein convertase, subtilisin/kexin type9, PCSK9)在低密度脂蛋白受体(low density lipoprotein receptor, LDLR)的代谢过程中起着重要的作用,因而间接影响到低密度脂蛋白胆固醇(low density lipoprotein cholesterol, LDL-C)水平。目前的研究已经证实,PCSK9功能获得突变可以急剧升高LDL-C水平,导致常染色体显性高胆固醇血症(autosomal dominant hypercholesterolemia, ADH)而PCSK9功能丢失突变能够降低血清LDL-C水平,降低冠心病(coronary artery disease, CAD)的发病风险；也有研究表明CAD患者中PCSK9水平升高会增加远期心血管事件的风险。因此,降低PCSK9水平从而实现对LDL-C水平的调控,是降脂治疗的新靶点。然而,在实现对PCSK9进行调控之前,有必要深入了解影响和调节PCSK9水平的因素,因为这是针对PCSK9治疗的前提和基础。研究表明PCSK9水平受到年龄,性别,饮食状态,激素水平等的调控。急性心肌梗死(acute myocardial infarction, AMI)是CAD最为严重的表现形式,其中伴随一系列病理生理改变,包括急性期反应,炎性细胞的激活和细胞因子的释放,血脂水平的改变。然而,作为调节血脂代谢的重要蛋白之一,PCSK9在AMI中的变化却鲜为人知,因此有必要探讨AMI对PCSK9的影响。此外,Krueppel样因子2(Krueppel-like factor2, KLF2)具有保护血管内皮和抗炎作用；有研究表明在动脉粥样硬化的患者中KLF2的表达量是降低的,但尚无研究报道在急性心肌梗死的情况下该因子的表达水平。目的：基于上述研究现状,本课题拟研究AMI情况下PCSK9水平的变化及其机制,PCSK9与血脂指标和炎症标记物的关系；同时探讨急性心肌梗死患者中KLF2的表达情况。方法：本研究分为临床研究和动物实验两部分。临床研究部分采用病例对照设计,由对照组(n=27),稳定型冠心病组(SCAD组,n=82)和AMI组(n=33)构成。AMI组患者从发病至就诊的时间间隔为≤12小时。抽取研究对象静脉血检测PCSK9,血脂指标和炎症标记物；利用淋巴细胞分离液分离外周血单个核细胞,提取RNA,进行荧光定量PCR测定外周血单个核细胞KLF2的表达。动物实验部分利用SD大鼠,通过结扎左冠状动脉前降支构建AMI模型,分别于手术后1小时,3小时,6小时,9小时,12小时和24小时处死大鼠,留取血标本和肝脏组织。血标本进行PCSK9,血脂指标的测定；肝脏组织提取RNA,进行荧光定量PCR,测定肝脏中PCSK9和调节PCSK9表达的核转录因子——固醇调节元件结合蛋白-2(sterol regulatory element-binding protein-2, SREBP-2)和肝细胞核因子1-a (hepatocyte nuclear factor1-α, HNF1-α)的表达。此外,同时测定受到SREBF-2调控的LDLR的表达。结果：AMI患者从发病至就诊时间的中位数为7小时。与对照组(340.56±77.62ng/mL)和SCAD组(334.99±85.96ng/mL)相比,AMI患者(290.42±79.05ng/mL) PCSK9水平明显降低(P值分别为0.01,0.02)。与对照组[中位数(第25及第75分位数)：0.37(0.30,0.46)]和SCAD组[0.39(0.32,0.5)]相比,AMI患者的FFA水平[0.5(0.41,0.72)]显著增高(P值均小于0.01)。AMI患者PCSK9与FFA呈负相关(r=-0.505,P=0.003),与其他血脂指标和炎症标记物无相关性。与对照组(4.50±5.70)和SCAD组(6.02±8.94)相比,AMI患者KLF2表达(2.81±0.92)无明显变化。动物实验结果显示AMI后早期(1小时至3小时)大鼠血清PCSK9的水平略有上升,随之下降(6小时至9小时),至12小时后上升,然后降低。荧光定量PCR结果发现AMI梗死12小时和24小时后PCSK9和LDLR mRNA升高；SREBP-2mRNA在AMI后24小时升高,而HNF1-α mRNA在AMI后6小时升高,12小时较假手术组无明显改变,24小时高于假手术组。结论：(1)AMI患者伴随PCSK9水平的升高,其表达的上调与核转录因子SREBP-2和HNF1-α有关；并且HNF1-α介导了早期(12小时内)PCSK9的升高而SREBP-2发挥作用主要在晚期(12小时之后)。(2)AMI状态下,PCSK9水平与FFA水平存在负相关,与其他血脂指标及炎症指标无相关性。(3)AMI发病过程中不伴有KLF2表达的变化。背景：他汀是目前降低低密度脂蛋白胆固醇(low density lipoprotein cholesterol, LDL-C)水平最为有效的治疗药物,是冠心病(coronary artery disease, CAD)—级和二级预防的基石。然而,增大他汀的剂量却并没有带来相应的LDL-C水平下降,长期以来,背后的原因并不清楚,直到近年发现他汀在降低LDL-C水平的同时,也升高了前蛋白转化酶枯草溶菌素9(proprotein convertase, subtilisin/kexin type9, PCSK9)的水平,而后者可以通过促进低密度脂蛋白受体(low density lipoprotein receptor, LDLR)的降解而减少LDL-C的清除,因而影响了他汀降脂的治疗效果。虽然临床研究表明他汀治疗4周或者更长时间均能升高PCSK9水平,但是有关短期应用他汀对PCSK9水平的影响却没有报道。依折麦布作为临床上广泛使用的另一类降脂药,在单独用药和与他汀联合用药降低LDL-C水平方面发挥着重要作用。然而,依折麦布是否也会对PCSK9水平有影响,目前的研究很少,且为横断面设计,缺少前瞻性随机对照试验的研究；关于依折麦布跟他汀联合用药对PCSK9的影响也无前瞻性研究。目的：基于上述研究现状,本课题拟研究(1)短期应用他汀,依折麦布及联合应用他汀和依折麦布对PCSK9水平的影响；(2)影响降脂药治疗后PCSK9水平变化的因素；(3)患者临床特征,血脂指标及超敏C-反应蛋白(high sensitive C-reactive protein, hs-CRP)与基线PCSK9的关系方法：本研究由两部分组成,第一部分纳入9例血脂异常的患者,给予匹伐他汀2mg/天治疗3天；第二部分纳入45例血脂异常患者,随机分为3组,分别给予依折麦布10mg/天(n=15),血脂康1200mg/天(n-15),依折麦布10mg/天+血脂康1200mg/天治疗3天(n=15)。分别于用药前和用药后第2天和第4天采血检测血清血脂指标,hs-CRP和PCSK9的浓度。采用spearman相关分析患者临床特征,血脂指标和hs-CRP与基线PCSK9的关系；采用多元线性逐步回归模型,分析患者临床特征,血脂指标,hs-CRP以及治疗药物对PCSK9治疗后变化百分率的影响。结果：匹伐他汀用药1天和3天分别升高了PCSK941%和27%(与用药前相比,P值均<0.05)；依折麦布用药1天和3天分别升高了PCSK929%和39%(与用药前相比,P值均<0.05)；血脂康用药1天和3天分别升高了PCSK932%和55%(与用药前相比,P值均<0.05)；依折麦布+血脂康用药1天和3天分别升高了PCSK936%和41%(与用药前相比,P值分别为<0.05和>0.05)。与单独用药相比,依折麦布+血脂康没有带来额外的PCSK9升高。LDL-C (r=0.356,P=0.008),总胆固醇(total cholesterol, TC)(r=0.376, P=0.005),高密度脂蛋白胆固醇(high density lipoprotein cholesterol, HDL-C)(r=0.283, P=0.038)和hs-CRP (r=0.334, P=0.014)与基线PCSK9水平呈正相关。多元线性回归显示,治疗前的LDL-C(p=-22.444,P=0.006)和甘油三酯(triglyceride, TG)(β=14.734, P=0.031)水平影响治疗后PCSK9变化百分率。结论：(1)匹伐他汀,依折麦布,血脂康以及依折麦布血脂康联合应用均可以在短期内升高PCSK9水平；与较单独用药相比,依折麦布血脂康联合用药并没有额外升高PCSK9水平。(2)基线LDL-C和TG水平可以预测降脂药治疗后PCSK9升高的幅度。(3)基线PCSK9水平与LDL-C, TC, HDL-C和hs-CRP呈正相关。更多还原

【Abstract】 Background:Proprotein convertase, subtilisin/kexin type9(PCSK9) plays an important role in the metabolism of low density lipoprotein receptor (LDLR) and hence indirectly impacts the low density lipoprotein cholesterol (LDL-C) levels. It had been proved that the PCSK9gain-of-function mutations could sharply increase the LDL-C levels and cause autosomal dominant hypercholesterolemia (ADH) while PCSK9loss-of-function mutations could decrease the LDL-C levels and associate with lower risk of coronary artery disease (CAD). Besides, study also indicated that CAD patients with high PCSK9levels were at high risk of long-term cardiovascular events. Hence, lowering PCSK9levels was the new target of managing the LDL-C levels. However, it is necessary to fully understand the regulatory factors involved in PCSK9before the manipulating of PCSK9, which is the foundation of forming a therapy targeting PCSK9. Studies had suggested that PCSK9levels were regulated by other factors such as age, gender, the status of dietary, and hormone. Acute myocardial infarction (AMI), the most severe form of CAD, was associated with a series of pathophysiological changes, including acute phase reaction, activation of inflammatory cells and release of cytokine, and changes in lipid levels. However, as one of the most important protein involved in lipid homeostasis, the change of PCSK9in the circumstance of AMI is unclear. Therefore it is necessary to explore its fluctuation in levels under AMI. In addition, studies had proved the endothelial protective and anti-inflammatory role of Krueppel-like factor2(KLF2) and its decreased levels were observed in patients with atherosclerosis. However, no study exists reporting its levels in AMI.Objective:Based on the aforementioned background, the present study was aimed to explore the change in PCSK9levels in AMI and the mechanisms behind it, the correlation between PCSK9and lipid parameters as well as inflammatory biomarkers, and the expression of KLF2under AMI.Methods:The present study consisted of clinical and animal parts. The clinical part used a case-control design, which included control group (n=27), stable coronary artery disease (SCAD) group (n=82), and AMI group (n=33). For patients with AMI, the time between symptom onset and hospital admission was less than12hours. Venous blood was drawn for all participants and PCSK9, lipid parameters and inflammatory biomarkers were measured. Peripheral blood mononuclear cells were isolated with Ficoll-Hypaque, their RNA was extracted and real time quantitative polymerase chain reaction (PCR) was used to measure the expression of KLF2. The animal part was conducted with SD rats. AMI model was constructed by ligation of the left anterior descending (LAD) branch of the coronary artery. The rats were killed at1,3,6,9,12, and24hours after the ligation of LAD, and blood samples and liver tissue were collected. The blood samples were used for the test of PCSK9and lipid parameters while liver tissue was used for RNA extraction. Real time quantitative PCR was performed to assay the expression of PCSK9and its regulatory nuclear transcription factors——(sterol regulatory element-binding protein-2, SREBP-2) and hepatocyte nuclear factorl-a (HNF1-α). In addition, the expression of LDLR, another target gene of SREBP-2, was also assayed.Results:The median time between symptom onset and hospital admission was7hours. Plasma levels of PCSK9were significantly lower in patients with AMI (290.42±79.05ng/ml) compared with those with SCAD (334.99±85.96ng/ml, P=0.01) and control subjects (340.56±77.62ng/ml, P=0.02). Plasma levels of free fatty acid (FFA) were significantly higher in patients with AMI [median (interquartile range):0.5(0.41,0.72)] compared with those with SCAD [0.37(0.30,0.46)] and control subjects [0.39(0.32,0.5)](both P<0.05). For patients with AMI, PCSK9levels were negatively correlated with FFA levels (r=-0.505, P=0.003), but no other association of other lipid parameters and inflammatory biomarkers with PCSK9was found. There was no significant difference in the relative expression of KLF2among patients with AMI (2.81±0.92), SCAD (6.02±8.94), and control subjects(4.50±5.70). The findings in animal experiment indicated that the levels of PCSK9slightly increased in the early stage (1-3hours) of AMI, followed by a decrease at6-9hours, an increase at12hours, and a decrease thereafter. Real time quantitative PCR results showed that the mRNA expression of PCSK9and LDLR increased at12and24hours after AMI and that SREBF-2mRNA increased at24hours after AMI, whereas HNF1-α mRNA increased at6hours after AMI with no significant change at12hours and increased levels at24hours when compared with sham group.Conclusion:(1) AMI was accompanied with high levels of PCSK9, the increased expression of which was mediated through SREBP-2and HNF1-α. The early (within12hours) up-regulation of PCSK9was mediated via HNF1-α whereas its late (after12hours) up-regulation was mediated via SREBP-2.(2) Under the circumstance of AMI, PCSK9levels was negatively correlated with FFA and on correlation exited between PCSK9and other lipid parameters and inflammatory biomarkers.(3) During the process of AMI, there was no significant change in the expression of KLF2. Background:Since statins are the most effective drugs used for lowering low density lipoprotein cholesterol (LDL-C), they are the cornerstone of the first and second prevention in coronary artery disease thus far. It had been observed for a long time that an increase in the dose of statins did not bring appropriate decrease in LDL-C. The mechanism behind this phenomenon was not clear until the findings that statins increase the levels of proprotein convertase, subtilisin/kexin type9(PCSK9) when decreasing the levels of LDL-C and PCSK9could promote the degradation of low density lipoprotein receptor (LDLR), which in turn negated the lipid-lowering effects of statins. Although clinical studies had proved that statin therapy for4weeks or longer would increase the levels of PCSK9, the short-term impact of statins on PCSK9has not been reported. Besides, ezetimibe, another class of lipid-lowering drug, which had also been widely used in clinical practice, plays a pivotal role in lowering LDL-C when in monotherapy or in combination with statins. However, studies concerning the impact of ezetimibe on PCSK9were limited and those published are cross-section design, hence there is a lack of prospective, random control study. In addition, no study exists reporting the impact on PCKS9of combination of ezetimibe and statin.Objective:Based on the aforementioned background, the present study was to investigate (1) the impacts on PCSK9of short-term statin, ezetimibe and the combined therapy of both drugs;(2) the factors associated with the percent change in PCSK9after lipid-lowering drugs therapy; and (3) the association of the clinical characteristics of patients, lipid parameters and high sensitive C-reactive protein (hs-CRP) with baseline PCSK9levels.Methods:The present study consisted of two parts with the first enrolling9patients with dyslipidemia and the second enrolling45patients with dyslipidemia. The patients in the first part were given pitavastatin2mg daily for three days, and those in the second part were evenly randomized into the following three groups:(1) ezetimibe group (ezetimibe10mg daily for three days);(2)xuezhikang group (xuezhikang1200mg daily for three days); and (3) combination group (ezetimibe10mg daily plus xuezhikang1200mg daily for three days). Fasting blood samples were collected before therapy and on the second and fourth day after the administration of drugs and the concentrations of lipid parameters, hs-CRP, and PCSK9in serum were measured. Spearman correlation analyses were performed to examine the association of the clinical characteristics of patients, lipid parameters and hs-CRP with PCSK9. Multivariate linear stepwise regression was used to explore the impacts on percent change in PCSK9of the characteristics of patients, lipid parameters, hs-CRP and drugs.Results:Pitavastatin increased PCSK9levels by41%at day1and27%at day3(both p<0.05compared with day0); ezetimibe increased PCSK9levels by29%at day1and39%at day3(both p<0.05compared with day0); xuezhikang increased PCSK9levels by32%at day1and55%at day3(both p<0.05compared with day0); ezetimibe plus xuezhikang increased PCSK9levels by36%at day1(p<0.05compared with day0) and41%(p>0.05compared with day0) at day3. LDL-C (r=0.356, P=0.008), total cholesterol (TC, r=0.376, P=0.005), high density lipoprotein cholesterol (HDL-C, r=0.283, P=0.038), and hs-CRP (r=0.334, P=0.014) were positively correlated with the baseline PCSK9levels. Multivariate linear regression indicated that baseline LDL-C (β=-22.444, P=0.006) and triglyceride (TG,β=14.734, P=0.031) levels were predictors of percent change in PCSK9after therapy.Conclusion:(1) Pitavastatin, ezetimibe, xuezhikang and the combination of ezetimibe and xuezhikang could increase PCSK9levels in short-term and combination of ezetimibe and xuezhikang did not result in greater increase in PCSK9compared to the monotherapy with either agent.(2) Baseline LDL-C and TG levels were predictors of percent change in PCSK9after therapy.(3) Baseline PCSK9levels were positively correlated with LDL-C, TC, HDL-C, and hs-CRP.更多还原