【作者】 吴焱贤；

【导师】 吴赛珠；

【作者基本信息】 南方医科大学 ， 心血管内科， 2013， 博士

【摘要】 一研究背景随着人民生活水平的提高以及人口老龄化进展的加快,近20年来我国心脑血管疾病发病率与死亡率逐年升高。冠心病(CAD)是由冠状动脉粥样硬化引起的心肌缺血性疾病,动脉粥样硬化(AS)是CAD的病理基础,脂质浸润学说贯穿了AS的形成、进展及恶化,并与其他学说密切相关,血脂异常是AS发生和发展的最重要因素之一。大量流行病学资料显示,AS的严重程度随血清胆固醇水平的升高呈正相关,血清胆固醇的浓度与冠心病的死亡率呈正相关。低密度脂蛋白胆固醇(LDL-C)升高导致的血脂异常是AS病理改变基础之一,AS,的严重程度与LDL-C水平的升高呈线性加重,LDL-C水平与各种心脑血管事件的发生率呈正相关。临床治疗上,我们采用他汀类等药物降低LDL-C水平,可显著减少心脑血管疾病的发生,降低LDL-C能让病人在临床治疗中获益。但是,尽管LDL-C水平达到指南要求甚至低于指南要求,但患者心脑血管事件发生率也只能降低30%左右,依然残留70%左右心血管事件发生率无法避免。高密度脂蛋白胆固醇(HDL-C)是另外一种重要的脂蛋白,HDL具有心血管保护作用,大量流行病学资料表明,血清HDL-C浓度与CAD发病危险呈独立的负相关；Framingham研究发现,血清HDL-C<35mg/dl者的CAD死亡率为血清HDL>55mg/dl者的4.1倍；血清HDL-C水平每升高1mg/d1,男性患者CAD的危险性即降低2%,女性降低3%。以上结果均提示,HDL是AS的保护因素之一,升高HDL-C水平是抗AS的靶点之一。HDL通过多种机制发挥抗AS的功效,包括对逆胆固醇转运(Reverse Cholesterol Transport, RCT)、抗氧化、抗炎、改善内皮细胞功能失调、抗血栓及促纤溶等。然而,越来越多的研究发现升高HDL-C水平并未达到人们预期的效果,CETP抑制剂torcetrapib尽管大幅度升高HDL-C水平,但torcetrapib组的全因死亡人数较阿托伐他汀组多,该研究不得不提前终止。Gomarasch等发现在载脂蛋白AI(ApoA-I)转变为ApoA-I Milano人群中,HDL-C的水平低,但其CAD的发生率也低且明显长寿。肝脏清道夫受体BI(SR-BI)高度表达小鼠,虽然降低了HDL-C水平,却具有显著的抗AS功能。因此HDL-C与AS的关系比我们想像中更加复杂,我们临床工作者及科研人员应重新审视HDL-C水平的意义,不单单需要关注HDL-C的水平,更需要关注HDL的功能对AS的影响。正常HDL功能的HDL才能发挥抗AS作用,而功能异常的HDL甚至有促AS进展的作用。HDL的多面性,与其结构复杂密切相关,HDL包含着多种酶和脂蛋白,其代谢途径也受多种酶的影响,其中任何一个脂蛋白性质的改变、酶的活性或者量的改变均能影响HDL的正常功能。RCT是HDL抗AS的重要的功能之一,RCT是一个复杂的过程,外周组织(包括动脉壁)游离胆固醇在多种酶的作用下与血清中ApoA-I结合后逐渐成为成熟的富含胆固醇的大颗粒HDL,其与肝脏SR-BI受体结合并被肝脏获取,最后HDL内的胆固醇通过肝细胞代谢,最终经胆汁排泄,从而使集聚在血管内皮下胆固醇排泄至体外,达到缓解甚至逆转AS的作用。RCT足由多种酶、多种受体、多种脂蛋白共同参与的复杂过程,ApoA-I,LCAT和CETP这两个酶在RCT过程中起着极其重要。抗炎抗氧化功能是HDL的另一个重要的功能。正常功能的HDL颗粒具有强大的抗氧化能力,一方面HDL含有多种抗炎抗氧化酶(如：PON1、ApoA-I),可以中和已形成的Ox-LDL的毒性反应；另一方面,HDL可以抑制氧化因子和炎症因子(如：MPO、LPO、SOD)的毒性作用,减少机体的炎症瀑布链反应,从而延缓AS的进展。正常情况下,ROS的产生和清除保持平衡状态,在动脉粥样硬化情况下,氧化产物生成大于抗氧化能力产生氧化应激,将导致血管的持续损伤,从而加剧动脉粥样硬化进展。功能异常的HDL容易形成Ox-HDL,而被氧化形成的Ox-HDL不单不具有抗氧化的能力,甚至促进Ox-LDL的形成。内皮的保护作用是HDL一个重要的功能。一方面HDL抑制单核细胞粘附和迁移到血管壁,刺激内皮细胞修复和增殖,抑制生长因子诱导的血管平滑肌细胞的增生；另一方面,HDL可以促进NOS催化内皮细胞内L-精氨酸产生,促进内皮细胞生成NO；升高血管内皮功能,还可以抑制血小板和白细胞在受损内膜的聚集和粘附,防止内皮功能受损。HDL可以通过多种途径促进eNOS的表达,减少抑制eNOS生成的因素,起到保护内皮功能的作用。脂蛋白相关磷脂酶A2(Lp-PLA2)是新近发现的与动脉粥样硬化密切相关,可将Ox-LDL水解成大量的溶血卵磷脂和游离氧化脂肪酸,这些促进动脉粥样硬化作用产物进一步活化粒细胞和Lp-PLA2,正常功能的HDL能结合Lp-PLA2,减少机体游离Lp-PLA2量,从而减少血栓的形成。Hcy升高同样也会引起多种疾病的发生,尤其增加心脑血管事件的发生的风险。同型半胱氨酸的合成和代谢保持着动态平衡,Hcy高于15.0μmol/L称为高同型半胱氨酸血症(hyperhomocy steinemia,高Hcy)。Hhcy是除高血压、高脂血症、吸烟、肥胖、心脑血管病家族史的一个新的独立的AS的危险因素,并且Hcy水平和血管危险之间呈正性相关关系。血浆Hcy每升高5μmol/L,缺血性心脏病发生率增加32%,卒中发生率增加59%,血浆Hcy每降低3μmol/L,可使缺血性心脏病风险降低11-20%,脑卒中风险降低15-33%。血浆总Hcy水平每升高5μmol/L则冠心病危险性男性增加60%,女性增加80%,相对危险性男性为1.6,女性为1.8,相当于总胆固醇每升高20mg/dL的危险性,因此高Hcy是冠心病的一个独立危险因素。脂质学说是动脉硬化的基础,目前国内外对Hcy与脂质学说的关系的研究主要集中在：1、Hey可以影响脂质代谢,使LDL代谢减慢,最终导致机体LDL-C水平的升高。2、Hey致LDL-C沉积作用,Hey可作用于低密度脂蛋白导致其载脂蛋白B的游离氨基巯基化,形成Hey与LDL复合物,这种被巯基化修饰的LDL可以不受负反馈抑制被巨噬细胞吞噬,胞内降解增加,引起胞内胆固醇堆积。3、Hcy致脂质氧化致炎作用：Hcy含自由巯基,可以自身氧化成Hcy混合性二硫化物和Hcy硫内酯,同时产生大量过氧化物,引起蛋白质损伤,清除氧自由基的酶活性降低,使得细胞和组织发生炎症反应,同时产生大量的Ox-LDL,加剧AS进展。HDL是心血管的保护因素之一,但其与Hcy的关系目前尚无明确的定论。Barter对近年HDL与HCY的关系进行综述发现,既有研究认为Hcy能影响HDL的功能,又有部分研究发现Hcy不影响HDL的功能。然而,越来越多的研究发现,Hcy可以通过影响HDL的某些酶的活性而影响HDL的功能,HCY通过影响PON1酶的活性,而影响HDL抗炎抗氧化功能。Clifford研究发现,HCY与CETP酶的活性密切相关,Hcy浓度升高可以影响CETP酶的浓度,而CETP是HDL代谢过程中的重要酶之一。然而HDL功能包括RCT功能、抗炎、抗氧化、维持细胞内皮稳定等功能,PON1和CETP仅仅是HDL众多功能酶中的一个,因此PON1活性和CETP酶并不能反映HDL的整体功能。Hcy通过影响PON1活性和CETP而调节HDL功能仅是其作用靶点之一,高Hcy可能通过更多的机制影响HDL功能。二研究目的本研究通过研究探讨Hcy和MTHFR基因与正常HDL-C患者冠状动脉硬化的关系,探讨Hcy与冠心病患者冠脉病变程度的相关性。通过检测高Hcy患者和对照组患者(两组患者HDL水平均在正常范围)HDL颗粒中的Apo-AI,LCAf、 CETP酶的含量,探讨高Hcy对HDL的RCT功能的影响。通过检测HDL颗粒中PON1、MPO活性,Lp-PLA2、LPO、SOD和eNOS水平,探讨高Hcy对HDL抗炎抗氧化能力的影响,从而为预防和治疗冠状动脉粥样硬化提供新靶点。三实验方法1.1研究患者入选标准、分组和排除标准本研究随机入选2011年1月～2012年6月佛山市顺德第一人民医院住院患者123例,入选患者全部采集吸烟史、高血压病史、糖尿病史、冠心病家族史和其他既往病史。根据根据美国心脏协会(AHA)推荐,正常空腹血浆同型半胱氨酸水平高于15.0μmol/L,则可诊断为高同型半胱氨酸血症,根据是否存在Hcy升高分为正常对照组和高Hcy组。两组入选人群的HDL-C均正常范围(1.16μmmol/L<HDL-C<1.55μmmol/L)。所有患者均签署知情同意书,如未取得知情同意,退出实验。排除标准：1、慢性肾功能不全患者、接受肾脏移植的患者；2、甲状腺功能亢进或甲状腺功能低下；3、肝功能受损或者既往存在肝脏病变患者；4、恶性贫血、急性白血病、癌症;5、目前或近期正口服甲氨蝶呤、茶碱、B族维生素及叶酸患者。根据入选标准、排除标准、符合HDL-C水平和是否签署知情同意书,有33例患者未纳入研究,共90例患者入选该研究,其中对照组35例,高HCY组55例。1.2血样的采集、保存和生化指标测定早晨8：00前抽取受试者空腹静脉血,部分静置离心放-80℃保存。部分血清及全血立即送顺德第一人民医院检验科用日立7600-210ISE型全自动生化分析仪测定测定：TC、TG、HDL-C、LDL-C、Apo-AI、Apo-B、FBS、PBS、Cr、 BUN、hs-CRP。1.3血浆Hcy的测定采用Agilent1100液相色谱仪,分别由不同的两人在同一天来测定,准确性和重复性均符合要求。批内变异为3.7%,批间变异为4.1%；质控样品测定结果偏差低水平点小于20%,中、高水平点测定结果偏差小于15%；样本两次测定值误差不超过15%；如果误差大于15%则重新测定,直到误差小于15%。1.5MTHFR基因型测定抽提空腹静脉血离心后得到的白细胞中的基因组DNA后,MTHFR C677T多态性位点基因型检测采用PCR-RFLP方法检测MTHFR基因分型,再通过2%琼脂糖凝胶电泳分开得到不同长度的片段。1.6冠脉病变情况所有患者均利用Philip H3000造影机进行冠状动脉造影采集影像,冠脉病变情况由2位专科医师仔细分析,意见有不一致时由第3人核实确定。按病变累及左前降支,左回旋支与右冠状动脉的支数,分为单支,双支和多支病变组；根据美国心脏协会规定的冠状动脉血管图像记录分段评价标准,采用Gensini积分系统对各支血管病变程度进行定量评定。1.7高密度脂蛋白的分离采用密度梯度离心法分离高密度脂蛋白,BCA法测定其浓度。1.8HDL逆转运酶的检测采用酶联免疫吸附剂盒测定HDL颗粒中的LCAT和CETP酶的量。1.9HDL抗炎抗氧化功能的测定采用酶联免疫吸附剂盒测定HDL颗粒中的eNOS和Lp-PLA2酶的量。采用分光光度计法测定PON1、MPO酶的活性和LPO、SOD的量。1.10统计学分析所有计量资料均以均数±标准差(x±s)表示,并采用SPSS13.0统计软件进行统计分析,计量资料采用两样本t检验,计量等级资料采用两独立样本秩和检验,计数资料采用例(%)表示,采用卡方检验,最后进行两等级变量间的Spearman相关性分析、两计数资料进行Pearson相关性分析和多元线性回归方法；P≤0.05为差异有统计学意义。四结果人口学特点比较与对照组相比(12.4±1.58mmol/L),高Hcy组(18.99±2.71mmol/L)患者的血清Hcy浓度明显升高(P=0.000),差异具有统计学意义。比较对照组和高Hcy组患者的年龄(χ2=0.407,P=0.685)、性别分布情况(χ2=0.003,P=0.959)、高血压患者(χ2=0.421,P=0.517)、糖尿病患者(χ2=0.778,P=0.378),吸烟患者(χ2=1.014,P=0.314)差异无统计学意义。两组患者的实验室指标比较比较对照组和高Hcy组患者的TC(t=0.534,P=0.595)、TG(t=0.457,P=0.649)、HDL-C (t=1.347, P=0.181)、LDL-C (t=0.650, P=0.517)、Apo-B (t=1.426,P=0.147)、空腹血糖(t=0.687,P=0.494)、餐后2h血糖(t=0.484,P=0.630)、Cr(t=0.301,P=0.764)、BUN(t=0.443,P=0.659)、ALT(t=0.716,P=0.476)、AST(t=1.725,P=0.088)间差异无统计学意义。入选两组患者生化指标情况一致,具有可比性。两组患者冠心病冠脉造影结果的比较对照组相冠脉多数无明显狭窄,或以单支病变为主,而高Hcy组患者冠脉病变程度严重,以单支和双支病变为主,对两组患者进行卡方检验发现,两组患者冠脉病变情况有差异,同时差异具有统计学意义(χ2=17.731,P=0.000)。再进行冠脉评分发现,高Hcy组患者冠脉积分情况较对照组升高,差异具有统计学意义(t=4.222,P=0.000)。两组患者MTHFR基因分型比较与对照组MTHFR基因分型以纯合型为主相比,高Hcy组患者MTHFR基因分型以杂合型的CT型为主,同时存在纯变异型TT型2例,进行卡方检验发现,两组患者分型有差异,同时差异具有统计学意义(χ2=7.463,P=0.024)。MTHFR基因分型与血浆Hcy水平及冠脉病变严重程度相关性分析血浆Hcy水平与MTHFR基因分型呈正相关(r=0.258,P=0.014),但相关关系并不密切。冠脉病变程度(r=0.600,P=0.000)和冠脉积分(r=0.621,P=0.000)与MTHFR基因分型呈正相关。Hcy水平与冠脉积分存在正相关(r=0.641,P=0.000)；Hcy水平与冠脉病变程度也呈正相关(r=0.438,P=0.000),但相关关系并不密切。最后,我们对冠脉积分与其他指标进行多元线性回归分析,以冠脉积分为因变量,其他变量为自变量,模型纳入MTHFR分型基因、HCY、TC、 Apo-AI水平模型有统计学意义(F=45.923,P=0.000)。MTHFR基因分型(t=7.851,P=0.000), HCY(t=5.805, P=0.000)、TC (t=2.748, P=0.007)、Apo-AI (t=-2.253,P=0.027)水平对冠脉积分有显著影响,差异具有统计学意义。高Hcy对Apo-AI的影响与对照组比较(1.27±0.12mmol/L),高Hcy组患者Apo-AI明显降低(1.21±0.13mmol/L)显著降低,差异均有统计学意义(t=2.28,P=0.025)。高Hcy对LCAT酶的影响与对照组比较(1029.02±133.88U/mg),高Hcy组患者LCAT酶(913.20±117.42U/mg)显著降低,差异均有统计学意义(t=4.318,P=0.000)。高Hcy对CETP酶的影响我们对HDL的重要代谢酶CETP检测发现,高Hcy组(26.33±4.13ug/mg)患者CETP酶比对照组(34.70±4.72ug/mg)明显降低,差异均有统计学意义(t=8.854,P=0.000)。高Hcy对PON1的影响与对照组比较(450.55±48.55U/ml),高Hcy组患者PON1活性明显降低(206.48±75.02U/ml)显著降低,差异均有统计学意义(t=17.087,P=0.000)。高Hcy对MPO酶的影响我们对MPO活性检测发现,高Hcy组(5.02+2.02U/L)患者MPO活性比对照组(3.15±1.49U/L)明显升高,差异均有统计学意义(t=4.737,P=0.000)。高Hcy对Lp-PLA2酶的影响与对照组比较(37.35±8.85ng/mg),高Hcy组患者Lp-PLA2酶明显降低(31.80±6.91ng/mg)显著降低,差异均有统计学意义(t=3.326,P=0.001)。高Hcy对LPO酶的影响与对照组比较(0.82±0.09umol/gprot),高Hcy组患者LPO量明显降低(0.93±0.9umol/gprot)显著升高,差异均有统计学意义(t=5.583,P=0.000)。高Hcy对SOD和eNOS的影响与对照组比较(190.32±44.57U/m1),高Hcy组患者SOD活性(204.30±49.37U/ml)升高,但差异无统计学意义(t=1.360,P=0.177)。我们再对eNOS活性检测发现,高Hcy组(5.02±2.02ug/mg)患者eNOS活性比对照组(3.15±1.49ug/mg)升高,但两者差异均无统计学意义(t=1.338,P=0.184)。五结论1.高Hcy组患者冠脉病变程度严重,冠脉积分情况较对照组升高,Hcy水平与冠脉积分存在正相关,提示高Hcy可能是冠心病的一独立危险因素。2.高Hcy组患者MTHFR基因分型以杂合型的CT型为主,同时存在少量纯变异型TT型；冠脉病变程度和冠脉积分呈与MTHFR基因分型呈正相关。3.高Hey可能通过降低HDL颗粒中Apo-AI量,降低LCAT、CETP酶量抑制HDL的RCT功能,促进动脉粥样硬化的进展。4.高Hcy通过降低PON1活性,减少HDL颗粒中Lp-PLA2量抑制HDL的抗炎抗氧化能力,促进动脉粥样硬化的进展。5.高Hcy通过升高MPO活性,增加HDL颗粒中LPO量而降低HDL的抗炎抗氧化功能,从而促进动脉粥样硬化的进展。6.高Hcy并不影响HDL颗粒中SOD和eNOS水平,该结果尚需进一步探讨。7.综上所述,高Hcy组患者冠脉病变程度严重,Hcy水平与冠脉积分呈正相关,提示高Hcy可能是冠心病的一独立危险因素,机制可能与Hcy患者HDL抗AS功能减弱(失功能)有关：高Hcy患者HDL颗粒中Apo-AI、 LCAT、CETP含量减少,抗炎抗氧化功能减弱,导致HDL的功能下降,促进动脉粥样硬化的进展。降低血浆Hey水平,可能改善HDL的功能,降低Hcy可能成为治疗动脉粥样硬化的靶点之一。更多还原

【Abstract】 Study BackgroundWith the improvement of people’s life quality and the rapidly aging population, the morbidity and mortality of cardiovascular and cerebrovascular diseases are increasing fast. Atherosclerosis (AS) is important pathological basis of coronary heart disease a myocardial ischemic disease cause by Coronary atherosclerosis. Dyslipidemia is one of the most important factors for atherosclerosis and it come with the formation, progress and deterioration of atherosclerosis. Almost all epidemiological studies had demonstrated a positive relationship between total cholesterol and low-density lipoprotein cholesterol whit mortality of cardiovascular. LDL-C level is the most important pathological changes of atherosclerotic risk factor. Patients can benefit from lowering LDL-C treatment through Statins. We found that patients’LDL-C level receiver desired level can reduce cardiovascular events by30%, but remain70%cardiovascular events would be happened. So we believe that high density lipoprotein (HDL) was a therapy target in atherosclerosis prevention.High-density lipoprotein cholesterol is another important lipoprotein which have Atheroprotective property. Almost all epidemiological studies had demonstrate an inverse relationship between plasma levels of HDL-C and cardiovascular atherosclerotic disease. The Framingham study found people of HDL-C levels of <35mg/dl have4.1times mortality rate than people of HDL-C levels of>55mg/dl Franceschini showed that for every1mg/dl increase in HDL-C, the predicted incidence of coronary events decreases by2%in men and3%in women. The major cardiovascular protective effects of HDL function may be attributed to its role in reverse cholesterol transport (RCT), anti-oxidant and anti-inflammation and so on.However, several lines of evidence indicate that the relationship between HDL and CAD risk is more complex and extends beyond the serum HDL-C levels. Torcetrapib, a potent CETP inhibitor, markedly increased the plasma concentration of HDL-C, but the risks of deaths and cardiac events in patients receiving tocetrapib had been increased simultaneously. The Milano people who carry the apolipoprotein A-I Milano mutant have very low serum HDL-C level while show very low incidence of CAD. Our results support the theory that not all HDL possess atheroprotective properties. Some HDL is dysfunctional or pro-inflammatory. Surem HDL-C level is not equivalent to HDL function.The proposed atheroprotective properties of HDL are multifaceted, including Reverse Cholesterol Transport and cholesterol efflux capacity, anti-oxidative and anti-inflammatory activities. The most important theory revolves around the role of HDL in macrophage reverse cholesterol rransport (RCT), in which excess cholesterol is effluxed to HDL and ultimately returned to the liver for metabolism by receptor of ATP binding cassette transporter A1(ABCA1) and Scavenger receptor class B type I (SR-BI).Structural modification and composition alteration of HDL may lead to HDL loss of normal biological function, and HDL-C is normal which still failed to inhibit atherosclerosis. The most important theory revolved around the role of HDL function is RCT, in which excess cholesterol effluxes to HDL and ultimately returned to the liver for metabolism. The process of RCT is extremely complicated:At first, cholesterol efflux from macrophages and then lipid-poor ApoA-I quickly acquires it via the ABCA1transporter. Lipidation of the lipid-poor ApoA-I and cholesterol complex generates nascent (pre-β) HDL. Subsequently, lecithin cholesterol acyl transferase (LCAT) mediated cholesterol esterification generates small HDL3particles; small HDL3can be converted to large mature HDL2in turn upon CETP [20]. At last, these mature HDL2transfer its cholesterol to the liver directly via SR-BI and subsequently excrete cholesterol through the bile. The process of RCT is extremely complex and ApoA-I, LCAT and CETP play an important role in this process.HDL has anti-inflammatory and anti-antioxidant property, which play an important role in AS protecting. HDL plays an important role in protecting against LDL oxidative modification, the enzyme PON1and ApoA-I contribute the key role to the antioxidative effects of HDL. MPO, LPO, SOD can change the structure of HDL which would oxidative modification HDL in to ox-HDL, and thus may reduce property of anti-inflammatory and anti-antioxidant in HDL, even to.produce more ox-LDL.The role of endothelium protection is an important function of HDL. On one hand, HDL inhibits monocyte adhesion and migration to the vessel wall; stimulate the repair and proliferation of endothelial cells, inhibited proliferation of vascufar smooth muscle cells by growth factor. On the other hand, HDL can promote endothelial cells producing the L-arginine and NO by-the NOS catalytic to elevated vascular endothelial function, can also prevent endothelial dysfunction by inhibit the aggregation and adhesion of platelets. HDL play a important role in the protecting endothelial function can by eNOS.Lipoprotein-associated phospholipase A2(LP-PLA2) is a newly discovered closely related with atherosclerosis. It hydrolyzed ox-LDL into a large number of lysolecithin and free oxidation of fatty acids. The function HDL can bind Lp-PLA2; reduce the free amount of Lp-PLA2and the formation of thrombus.High homocysteine causes many kinds of diseases, especially increase the risk of cardiovascular and cerebrovascular events. The synthesis and metabolism of homocysteine are maintaining homeostasis, higher than15.0μmol/L called high hyperhomocysteinemia (HHcy). Hhcy is a new independent risk factor of AS including hypertension, hyperlipidemia, smoking, obesity, cardiovascular and cerebrovascular disease, which was a positive relationship between Hcy levels and cardiovascular risk. The plasma Hcy escalate5umol/L can increase ischemic heart disease by32%and stroke by59%; Plasma Hcy reduced3umol/L, the risk of ischemic heart disease can be reduced by11to20%, and the risk of stroke reduced by15-33%. Total plasma Hey level is increased by5umol/L is equivalent to the the total cholesterol each elevated a20mg/dL risk, and therefore to be an independent risk factor for coronary heart disease.Dyslipidemia is the basis of atherosclerosis. Relationship of Hey and dyslipidemia are unclear. HHcy slows down LDL metabolism and elevated levels of LDL-C. Hey thiolated low density lipoprotein into Hcy-LDL complexes, which cannot be macrophage phagocytosis, increased intracellular degradation, caused by the intracellular accumulation of cholesterol. HHcy can autoxidation to produce large amounts of peroxide, causing protein damage, which produce large amounts of Ox-LDL, exacerbated atherosclerosis progression.High-density lipoprotein is cardiovascular protective factor, but its relationship with Hey is no clear now. A research found that Oral folic acid to reduce high Hey can be improved PON1activity in the HDL particles, thereby reducing the content of diabetes Ox-LDL and alleviate the progression of atherosclerosis. PON1is one of the HDL enzymes; PON1can not reflect the HDL function. HHcy may affect HDL function through more mechanism.ObjectivesThe study was to investigate the relationship between HCY and MTHFR gene and atherosclerosis in in Patients with Medium HDL level, explore the relevance of HCY with coronary artery disease severity. We investigated the impact of HHcy to the RCT function of HDL by detecting the amount of Apo-AI, of LCAT, CETP in HDL particles. We also investigated the anti-inflammatory and anti-antioxidant function of HDL by detecting the PONl and MPO activity, the amount of Apo-AI, of LCAT, CETP in HDL particles.Methods1. Patient inclusion criteria, grouping and exclusion criteriaWe collect the patient’s history of smoking, hypertension, diabetes, family history of coronary heart disease and other past medical history. We divided patients into two groups according HCY levels the HHcy group was defined HCY level above15.0μmol/L and the control group was below15.0μmol/L (guideline of ACC). Both two group HDL level was normal (40mg/dL <HDL-C <60mg/dL). All patients signed informed consent, who exit experiment without obtaining the informed consent. Exclusion criteria:1, chronic renal insufficiency patients, patients receiving kidney transplants;2, hyperthyroidism or thyroid dysfunction;3, impaired liver function or pre-existing liver disease patients;4, pernicious anemia, acute leukemia, cancer;5, the currentor recent oral methotrexate, theophylline, B vitamins and folic acid in patients.2. Serum lipid analysisBlood lipid analysis was performed at the beginning of the experiment. Serum triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (HDL-C) concentrations were measured by using an automated biochemical analyzer.3. HCY analysisHCY analyses were using Agilentl100liquid chromatography, respectively determine by two people in the same day. Of variation of3.7%, inter-assay variation was4.1%; deviation of measurement results of the quality control samples of low-level point of less than20%deviation of the measurement results, a high level point of less than15%; the sample twice determination error does not exceed15%; if the error is greater than15%is re-measured, until the error is less than15%.4. MTHFR gene analysisMTHFR was test by PCR-RFLP, and through2%agarose gel electrophoresis to separate the different length fragments5. Coronary lesionsCoronary angiography was carry by Philip H3000. Coronary lesions were divided into single, double and multi-vessel disease. Gensini score system quantitates assessment of severity of vessel:narrow <25%1score,25%-49%2score,50%-74%4score,75%-90%8score,91%-99%16score,100%32score.6. The separation of high-density lipoproteinHDL separated by density gradient centrifugation. BCA assay was use to analysis the HDL particles concentration7. Assessment of the RCT function of HDLLCAT and CETP were assayed using Elisa.8. Assessment of the anti-inflammatory and anti-antioxidant function of HDLPON1, MPO, LPO, SOD in HDL particles was assayed using kits according to the manufacturer’s instructions. eNOS and Lp-PLA2were assayed using Elisa.Results1.There were no differences in Age(χ2=0.407, P=0.685), sex (χ2=0.003, P=0.959), hypertension(χ2=0.421,P=0.517), diabetes(χ2=0.778, P=0.378), smoking (χ2=1.014, P=0.314), TC (t=0.534, P=0.595)、TG (t=0.457, P=0.649) HDL-C (t=1.347, P=0.181)、LDL-C (t=0.650, P=0.517)、Apo-B (t=1.426, P=0.147)、FBG (t=0.687, P=0.494)、PBG (t=0.484, P=0.630)、Cr (t=0.301, P=0.764)、BUN (t=0.443, P=0.659)、ALT (t=0.716, P=0.476)、AST (t=1.725, P=0.088) in two group,2. The Coronary lesions were Serious in HHCY group than control group (χ2=17.731, P=0.000).The Gensini score was higher in HHCY group than control group (t=4.222, P=0.000)3. There were significant differences in two group in MTHFR gene (χ2=7.463, P=0.024). HCY levels were positively correlated with MTHFR (r=0.258, P=0.014), Coronary lesions(r=0.438, P=0.000) and Gensini score(r=0.641, P=0.000). MTHFR gene were positively correlated with Coronary lesions(r=0.600, P=0.000)and Gensini score(r=0.621, P=0.000).Finally, we take multiple linear regression analysis our data, we found that Gensini score were closed correlated with MTHFR gene (t=7.851, P=0.000), HCY (t=5.805, P=0.000)、TC (t=2.748, P=0.007)、Apo-AI (t=-2.253, P=0.027), the model was meaning full (F=45.923, P=0.000)4. Conpared with control group, Apo-AI (1.27±0.12mmol/L vs1.21±0.13mmol/L,P=0.025), LCAT (1029.02Q133.88U/mg vs913.20±117.42U/mg, P=0.000) and CETP (34.70±4.72ug/mg vs26.33±4.13ug/mg,P=0.000) was significantly lower in HHCY group.5. Conpared with control group, PON1(450.55±48.55U/ml vs206.48±75.02U/ml, P=0.000) and Lp-LPA2(37.35±8.85ng/mg vs31.80±6.91ng/mg, P=0.001) was significantly lower in HHCY group.6. Conpared with control group, MPO (3.15±1.49U/L vs5.02±2.02U/L, P=0.000) and LPO (0.82±0.09umol/gprot vs0.93±0.9umol/gprot, P=0.000) was significantly higher in HHCY group.7. There were no differences in eNOS and SOD levels.Conclusions1. HHCY group coronary lesions were serious than control group, and Gensini score were higher than control group. The HCY level was positive correlated with Gensini score.2. MTHFR gene in HHCY group was significant differences with control group. The MTHFR gene was positive correlated with Gensini score and coronary lesions. High Hcy may be an independent risk factor for coronary heart disease.3. HHCY may reduced the RCT function of HDL through decreased. Apo-AI, LCAT, CETP in HDL particles.4. HHCY reduced the anti-inflammatory and anti-antioxidant function of HDL through decreased PON1and Lp-PLA2in HDL particles.5. HHCY reduced the anti-inflammatory and anti-antioxidant function of HDL through increased MPO and LPO in HDL particles.6. HHCY were no effect of SOD and eNOS of HDL, the result need more research to confirm.7. All of above, High Hcy may be an independent risk factor for coronary heart disease. HHCY reduce the RCT, anti-inflammatory and anti-antioxidant function of HDL to aggravate atherosclerosis. Reduce plasma homocysteine levels may improve the function of HDL, and reduce Hcy may become one of the targets for the treatment of atherosclerosis.更多还原