【作者】 费瑜；

【导师】 李淑梅；

【作者基本信息】 吉林大学 ， 内科学， 2011， 博士

【摘要】 AF是临床最为常见的心律失常之一。据流行病学调查显示,近年来AF的发病率有逐年上升趋势,年龄越大发病率越高,且有器质性心脏病者AF发病率高达40%。AF的危害不仅在于其发作时的临床症状会严重影响患者的生活质量,而且还在于其较高的血栓栓塞发生率导致致残和致死率显著增加。因此,探讨其发病机制并对其加以防治一直是人们关注的热点。近年来,有关AF发生机制的研究已取得较大进展,现认为AF的始发和维持是多种机制共同参与的结果。尽管多种因素相互作用,使得AF的发生变得更为复杂,但归结起来主要涉及两个方面：一是触发因素,表现在刺激交感神经和迷走神经,引起心动过缓、房性期前收缩、房性心动过速和房室传导阻滞等对心房的牵拉作用,使心房压力变化,导致AF的发生；二是结构重构和电重构,这些重构有利于折返的形成。AF电重构主要包括心房ERP和APD缩短,AP传导速度减慢,ERP离散度增加等电生理特征的改变,这种改变有利于AF的发生和维持。电重构的基础是心房肌细胞离子通道和跨膜离子流的改变。心房结构重构在细胞水平上主要体现在两方面：一是心房肌细胞超微结构的改变,包括心房肌细胞退行性改变、内质网扩张、线粒体肿胀、嵴的消失、闰盘非特化区增宽以及糖原颗粒减少和肌原纤维增加；二是心房肌间质发生变化,表现在心房肌间质纤维增生、间质纤维化和心房增大,使得电传导不一致,有助于局部传导阻滞或折返的形成,引起AF的发生。在分子水平上的变化表现为结构蛋白、收缩蛋白、缝隙连接蛋白、离子通道蛋白的降解,使得心房肌细胞的连接和信号传导异常,导致AF的发生和维持。业已发现,OFR、基因的突变、自主神经功能紊乱、离子通道异常和炎症反应等均与AF的发生和维持有关,但炎症与AF的关系越来越引起人们重视。1997年Bruins等首次报道了CRP和IL-6在AF发生时有明显升高的变化,随后有学者在进一步证明CRP与AF关系的同时,还发现其它炎症因子如IL-8、TNF-α等也与AF的发生和发展有关,这些实验结果一方面确立炎症反应在AF发病过程中的地位；另一方面说明炎症可能是AF发生的独立因素,是导致AF的原因。IL-10是1989年Fiorentio从小鼠Th2细胞上清液中发现的一种蛋白,它能抑制Th1的功能,因而被称为细胞因子合成抑制因子。研究表明,IL-10能抑制活化的T淋巴细胞、自然杀伤细胞和巨噬细胞产生细胞因子,能抑制单核巨噬细胞表面主要组织相容抗原Ⅱ类分子的表达,降低抗原提呈细胞的抗原递呈能力,能抑制大鼠AMI后左心室胶原的沉积,改善心室重构,因而它在下调炎症反应和抑制心脏重构中发挥重要作用。近年来,人们发现一种新的炎症标记物—PTX3,并已证明在冠心病、急性冠脉综合征时其水平有明显升高,且与不稳定型心绞痛危险分层有关。在与急性冠脉综合征的相关性研究中,PTX3明显要优于CRP,但PTX3在AF发生和发展中的作用及它与IL-10的关系目前所知甚少,故本文对AF患者心房肌细胞IL-10和PTX3进行研究,旨在揭示它们在AF中的作用,为炎症与AF关系的理论奠定实验基础。目的：探讨PTX3和IL-10在AF发生和维持中的作用及机制,为进一步揭示炎症与AF的关系,防止AF的发生和对其积极加以治疗提供实验依据。方法：1研究对象：根据《赫尔辛基宣言》精神,所有患者术前均经伦理委员会讨论,并已征得患者及其家属知情同意。选择行心脏外科手术患者22例, AF组11例,男6例,女5例,年龄41.5±4.3岁,心功能Ⅱ级9例,Ⅲ级2例,入选患者无其它心律失常病史且AF持续时间大于1年。SR组11例,男5例,女6例,年龄40.7±53岁,心功能Ⅱ级8例,Ⅲ级3例,入选患者无心律失常病史。两组均记录年龄、性别、心功能NYHA分级、心电图、超声心动图及有关实验室检查。甲状腺机能亢进,扩张性心肌病、慢性肺源性心脏病、感染的急性期、肥胖、风湿性疾病、血液和造血系统疾病、内分泌和代谢性疾病、严重肝肾功能不全、近期应用抗生素、阿斯匹林(术前7天停用除外)、ACEI和ARB、他汀类调脂药物等均不列入本研究范围之内。2标本采集：心脏手术中建立体外循环,于心脏停跳前获取右心耳500mg,除去血液和脂肪组织,将其中200mg置入液氮瓶中,然后送入-70℃深低温冰箱冻存、备用。用PCR法和WB法观察慢性AF患者心房肌细胞IL-10和PTX3的变化；另300mg用10 ml氧饱和的无钙液在37℃保温下,于5-10 min内送到实验室。分离心房肌细胞,首先,将AF组分成8份,在4份中,有3份分别加入浓度为8μg/ml、10μg/ml、12μg/ml的PTX3各1ml,与余下的1份和SR组一起放入恒温箱中,作用1h,用PCR法和WB法观察KAch基因和蛋白的变化；另4份做成膜片后,其中3份在灌注液中加入不同浓度的PTX3各1ml,作用20min,用膜片钳技术测定IKAch密度。另外,将AF组分成两份,其中1份加入1ml浓度为12μg/ml的PTX3,与余下1份和SR组一起放入恒温箱中,作用1h,收集上清液,用PCR法、WB法和ELSIA法观察IL-6、TNF-α的水平和NF-κB基因及蛋白的变化。最后,将AF组分成4份,做成膜片后,其中1份加入1m1浓度为12gg/ml的PTX3和1ml浓度为10μg/ml的IL-10,另两份分别加入12μg/ml的PTX3和10μg/ml的IL-10各1ml,用膜片钳技术观察IKAch密度的变化。结果：1慢性AF患者心房肌细胞IL-10 mRNA及IL-10蛋白表达明显低于SR组,而PTX3mRNA及蛋白表达明显高于SR组,IL-10mRNA表达与PTX3 mRNA表达呈负相关,IL-10蛋白表达与PTX3蛋白表达呈负相关,统计学均有显著差异。2 AF组的Kir3.4mRNA表达、GIRK4表达及IKAch密度明显低于SR组,PTX3的剂量越大,它们降低越明显,统计学均有显著差异。3心房肌细胞NF-κBP65的mRNA及蛋白表达和IL-6及TNF-α的表达水平在AF组明显高于SR组,其中大剂量组明显高于AF组,且NF-κBP65的表达分别与IL-6和TNF-α的水平呈正相关,统计学均有显著差异。4大剂量组+IL-10的IKAch密度明显高于大剂量组,AF组+IL-10明显高于AF组和大剂量组+IL-10,统计学均有显著差别。结论：1IL-10和PTX3参与了AF的发生和维持。2 PTX3可通过对慢性AF患者心房肌细胞Kir3.4mRNA表达、GIRK4表达和IKAch密度的影响,使得AF发生和维持。3 PTX3可通过对慢性AF患者心房肌细胞NF-κB、IL-6和TNF-α的影响,促进炎症反应,引起心房重构。4 IL-10在一定程度上对AF的炎症反应有抑制作用,可用于防治AF的心房重构。更多还原

【Abstract】 Atrial fibrillation (AF) is one of the commonest clinical arrhythmia. According to epidemic disease survey, AF’s morbidity has been an increasing trend for the past few years, the older people are, the higher the morbidity is; people of organic cardiopathy even have a morbidity up to 40%. Not only would AF seriously influence life quality in paroxysmal clinical symptoms, but also the comparative high incidence of its thromboembolism would cause obvious increase of cripple and death. Thus, it has been a focus of people to pay attention to probing into its pathogenesis and preventive treatment.Recently, great progress has been made on AF mechanism, It is considered that the origin and maintenance of AF is the result of a variety of mechanisms to participate, despite the interaction of multiple factors in the pathogenesis of AF makes it extremely complicated, but which mainly refers to 2 aspects:The first is triggering factors, which is reflected in stimulus between sympathetic nerve and vagus nerve, leading to tractive function of bradycardia, atrial premature contraction, atrial tachycardia and ventricular block towards atrium cordis, change pressure of atrium cordis, then AF occurs; the second is structure reconstitution and electricity reconstitution, which are good for forming fold-back. AF electricity reconstitution mainly includes time limit shortening of ERP and AP of atrium cordis, AP’s conduction speed decreases, ERP’s divergence factor increases change of isoelectric physiological feature, which are good for occurrence and maintenance of AF, foundation of electricity reconstitution is change of cardiac atrium myocytes ion channel and transmembrane ion flow. Reconstitution of atrium cordis structure is mainly reflected in 2 aspects:The first is change of cardiac atrium myocytes ultrastructure, including regressed nature change of cardiac atrium myocytes, mitochondria swell, iliac crest disappearance, unspecialized region broadening of intercalated disc, glycogenosome decrease and myofibril increase; the second is change of cardiac atrium mesenchyme, which is shown in hyperplasia of cardiac atrium interstitialfibers, atrium cordis accretion, inconsistent electricity conduction by mesenchyme fibrosis, these are good for partial conduction retardant or fold-back and causes occurrence of AF. While changes on molecular level is shown in degradation of structural proteins and contractile proteins, disorganized gap junction proteins and degradation of ion channel proteins, which lead to abnormality of cardiac atrium myocytes junction and signal conduction and cause occurrence and maintenance of AF.It has been found in the field that OFR, genovariation, autonomic nerves dysfunction, ion channel abnormal and inflammation reaction have all relationship with occurrence and maintenance of AF, while relation between inflammation and AF has been increasingly valued by people. Obviously increase changes of CRP and IL-6 when AF occurred were reported by Bruins etc in 1997 for the first time, soon afterwards scholars further proved that AF’s occurrence and development had relation with not only CRP but also other inflammatory factors as IL-8, TNF-αetc; on one hand, all the experimental results have established position of inflammation in AF, on the other hand, they have indicated that inflammation is a potential independent factor and reason for occurrence of AF.IL-10 is a kind of albumen found in Th2 cell supernatants fluid of little mouse by Fiorentio in 1989, which can restrict function of Th1 and is called composition and inhibiting factor of cell factor as a result. IL-10 can restrict activated T lymphocyte、natural killer cell and macrophage from generating cell factor, and expression of main compatible organ antigen II molecule on surface of mononuclear phagocyte and reduce antigens submission capacity of antigen presenting cell, besides, it has been found that IL-10 can restrict ventriculus sinister collagen deposit after myocardial infarction of big rat and improve reconstitution of heart ventricle, thus, it plays important role in regulating inflammation reaction and restrain heart reconstitution. For the past few years, a kind of new inflammation marker-PTX3 has been found, whose level has been proved obviously rising in coronary heart disease, acute coronary syndrome and has relation with danger layering of unstable angina pectoris. Relativity to acute coronary syndrome, plasma PTX3 level exceed CRP. However, PTX3’s function in AF’s occurrence and development and its relation with IL-10 have been rarely known at present, thus, IL-10 and PTX3 of cardiac atrium myocytes for AF patients are studied in the thesis, aiming to reveal their functions in AF and offer experimental basis for inflammation and AF theory.Purpose:To discuss function and mechanism of PTX3 and IL-10 in AF’s occurrence and maintenance and to offer experimental grounds for further prompting relation between inflammation and AF, prevent occurrence of AF and cure it positively.Method:1 Research object:According to Declaration of Helsinki,22 patients suffering cardiac surgical operation are selected after gaining preoperational discussion of Ethics Committee and permission of patients and family members,11 in AF group,6 men and 5 women with age of 41.5±4.3 years,9 patients haveⅡdegree heart function,2 patients haveⅢdegree heart function, no additional arrhythmia medical history,AF time is over 1 year.11 in SR group,5 men and 6 women with age of 40.7±5.3 years.8 patients haveⅡdegree heart function,3 patients haveⅢdegree heart function, no arrhythmia medical history. Age, gender, heart function NYHA grade, electrocardiogram, ultrasonic cardiogram and related laboratory examination are all recorded in the two groups. Hyperthyroidism, dilated cardiomyopathy, chronic pulmonary heart disease, acute infection, fat, rheumatic disease, blood and hemopoietic system disease, internal secretion and metabolic disease, serious liver dysfunction, recent application of antibiotics, aspirin (exclusive of disuse 7 days before operation), ACEI and ARB, statins adjustment lipid deugs are all not listed in this research scope.2 Sample collection:Extracorporeal circulation should be established in the cardiac surgery and 500mg auricula dextra before cardiac arrest should be acquired, removing blood and adipose tissue, among which 200mg is imbedded with liquid nitrogen, input and frozen in -70℃profound hypothermia refrigerator for reservation. observe changes of cardiac atrium myocytes IL-10 and PTX3 of chronic AF patients by PCR method and WB method. On top of 300mg,Put 10ml oxygen-saturation non-calcium liquor under 37℃preservation and send it to the laboratory within 5-10 minutes. In separation of cardiac atrium myocytes, firstly, becomes the AF component 8, in 4, some 3 join separately concertration on 8μg/ml, 10μg/ml.12μg/ml PTX3 each lml, with in addition 1 and the SR group together puts in the thermostat,affects 1h, observes the KAch gene and protein change by PCR method and WB method; After another 4 make the patch,3 join the different concertration PTX3 each 1ml in the perfusate, affects 20min, determines the IKAch density by the patch clamp technique. Moreover, becomes the AF component 2,1 joins the lml concertration 12μg/ml PTX3, with addition 1 and the SR group puts in the thermostat together, affects 1h, the collection supernate, by PCR method、WB method and the ELISA method observes IL-6、TNF-αlevel and NF-κB gene and protein change. Finally, becomes the AF component 4, after making the patch,1 joins the 1ml concertration 12μg/ml PTX3 and 1ml concertration 10μg/ml IL-10, another two join separately 12μg/ml PTX3 and 10μg/ml IL-10 each 1ml, observes the IKAch density by the patch clamp technique.Results:1 IL-10 mRNA and IL-10 protein expression of cardiac atrium myocytes of chronic AF patients are obviously lower than SR group, while PTX3mRNA and PTX3 expression are obviously higher than SR group, Both expression of IL-10 and PTX3 take on negative correlation. significant differences in statistics exist in both groups.2,expression of Kir3.4mRNA and GIRK4 and IKAch density in AF group are obviously lower than SR group, the more increase PTX3 dose it is,the lower there are, significant differences in statistics also exist in all groups.3, NF-κBP65 mRNA and protein expression of AF group and Supernate IL-6 and TNF-αlevel of AF cardiac atrium myocytes are obviously higher than SR group, those of the large dose group are obviously higher than AF group, NF-κBP65 takes on positive correlation with IL-6 and TNF-α,significant differences in statistics also exist in all groups.4, IKAch density of large dose group +IL-10 is obviously higher than the large dose group, AF group +IL-10 is higher than AF group and large dose group + IL-10, significant differences in statistics also exist in all groups.Conclusions:1 IL-10 and PTX3 participates in occurrence and maintenance of AF.2 PTX3 can make AF occurrence and maintain,by means of influencing on current density of KAch, expression of Kir3.4mRNA and GIRK4 protein.3 PTX3 can promotes inflammation reaction and cause restructure of atrium cordis, by means of influencing on NF-κB, IL-6 and TNF-αof cardiac atrium myocytes of chronic AF patients.4 IL-10 can restrict AF inflammation reaction to some extent, and restrict in restructuring AF atrium cordis.更多还原