【作者】 黎健勇；

【导师】 吴平生； 吴书林；

【作者基本信息】 南方医科大学 ， 心血管内科， 2013， 博士

【摘要】 随着人口老龄化及平均寿命延长,心房颤动(Atrial Fibrillation, AF)的发病率越来越高,其对人群死亡率、致残率的影响非常显著。预计2020年我国人口将达到14.5亿,60岁以上老年人约占人口总数20%,医务工作者面临艰巨的房颤预防和治疗工作。近年心房颤动研究进展迅速,药物治疗和导管射频消融术取得了一定进步,但许多患者治疗效果不佳,因此必须进一步深入研究其发病机制,寻求新的治疗途径。房颤的始动因素很多,随着房颤持续时间延长,心房电生理和细胞特性发生改变使房颤得以持续,这一过程称为电重构,主要特点包括动作电位时限(APD)和心房有效不应期(ERP)缩短及离散度增加,频率适应性降低,心房传导速度减慢使心房波长缩,使房颤易于诱发及稳定性增加。而心房不应期和动作电位的缩短,与内向离子流(Ca2+或Na+)净减少,外向离子流(K+)净增加,缝隙连接减少密切相关。心房电重构发生的基础就是离子通道的损伤或者功能改变,其机制复杂,早期研究认为心钠素、心房肌缺血与ATP敏感性钾通道、自主神经系统、钙超载和代谢紊乱可能是房颤电重构的触点,近年来人们将注意力集中于氧化应激和损伤的途径上。多项研究证实AF患者右心耳组织、血浆中的高敏C反应蛋白(hs-CRP)、肿瘤坏死因子(TNF-α)、白介素-6(IL-6)较窦性心律组明显升高,TNF-α血清浓度在阵发性、持续性和永久性房颤中呈进行性递增,认为氧化应激和炎症可直接导致房颤的病理生理改变。动物实验发现TNF-α可影响到大鼠心室肌L型钙电流、CX40,对其它离子通道及缝隙链接作用不明确。炎症因子TNF-α IL-6、IL-4参与激活和调节JAK-STAT信号通路,JAK-STAT信号通路可上调环氧化酶(COX-2)、一氧化氮合酶(NOS2)、血管内皮生长因子(VEGF)、抗氧化剂锰超氧化物歧化酶(MnSOD)、金属硫蛋白(MT1a nd MT2)、基质金属蛋白酶(MMP)等,从而影响心肌肥厚和心肌重构。研究发现藜芦醇通过阻断JAK/STAT1通路从而达到控制干扰素介导的巨噬细胞迁徙,能降低炎症反应对血管的损害,降低动脉血栓和斑块的形成。在STAT3基因敲除小鼠中发现心脏间质纤维化增加,TNF-α分泌增多,心肌扩张和心功能受损；敲除STAT3基因或者使用JAK2抑制剂AG490降低STAT3表达,可使心肌梗死面积增大；Adawi研究发现大鼠心肌梗死模型中,予AG490后Kv4.3mRNA表达增加,可见JAK-STAT信号通路是参与了多种心血管疾病的病理生理。本研究通过观察TNF-α慢性刺激HL-1细胞,探讨TNF-α对部分离子通道及缝隙连接的影响,JAK-STAT信号通路在炎症因子TNF-α作用下,参与调节电重构的机制。本实验分两部分。第一部分TNF-α对HL-1心房肌细胞电重构的影响目的：TNF-α参与冠心病、心力衰竭、风湿性心脏病、先天性心脏病的多种心血管疾病病理过程,但在房颤发病机制中的作用尚不清楚。本部分实验通过不同浓度TNF-α刺激HL-1细胞后,通过检测电重构相关的基因,探讨TNF-α对HL-1细胞电重构的影响。方法：TNF-α25ng/ml、50ng/ml刺激HL-1细胞24小时后,提取总RNA和总蛋白,使用Real-time PCR(RT-PCR)检测CACNA1c、KCNJ2、KCNH2、KCNQ1、 GJA5、GJA1、SLC8A1的mRNA表达,用蛋白印迹法(Western bloting)测定CAV1.2和ERG的表达,并用膜片钳方法记录对照组和实验组Ica,L、IKR电流和APD。采用pCLAMP10.2软件对单个全细胞记录进行数据和图形转换,Prism3.0软件对数据进行曲线拟合及绘制离子通道电流密度及APD,电流密度(pA/pF)=电流强度/电容。所有数据用均数±标准差(Mean±SD)表示,mRNA比较采用one-way ANO VA方法检验,采用LSD方法进行两两比较。IcaL、Ikr、 APD采用两独立样本t检验。用SPSS13.0统计软件分析,以P<0.05为有统计学意义。结果：RT-PCR发现1、CACNAlc mRNA水平随着TNF-a浓度的增加而减少,对照组与实验组有显著差异(p<0.01),而实验组TNF-a50ng/ml组也明显低于25ng/ml (p<0.01)。2、KCNJ2mRNA水平随着TNF-a浓度的增加而增加,但对照组与25ng/ml组无明显差别,与50ng/ml有显著差异(p<0.05),实验组之间无显著差别。3、KCNH2mRNA实验组显著高于对照组(p<0.05),但实验组之间无显著差别。4、KCNQ1mRNA实验组与对照组之间无显著差异。5、GJA5mRNA实验组与对照组之间无显著差异。6、GJA1mRNA水平随着TNF-a浓度的增加而增加,但对照组与25ng/ml组无明显差别,对照组、25ng/ml组与50ng/ml有显著差异(p<0.05)。7、SLC8A1mRNA水平随着TNF-a浓度的增加而增加,对照组与25ng/ml组、50ng/ml组有显著差异(p<0.05和p<0.01),实验组25ng/ml组与50ng/ml之间也有显著差异。蛋白印迹法发现钙离子通道CAV1.2随着TNF-a浓度增加而减少,钾离子通道ERG随着TNF-a增加而增加。通过膜片钳检测发现与对照组相比,TNF-a组L型钙通道离子电流密度减少,电流密度在指令电位-10mv、0mv、10mv、20mv、30mv、40mv明显下降有显著差。与对照组相比,TNF-α组快速激活延迟整流钾电流平台期和尾电流的电流密度增加,平台期电流密度在指令电位-40、-30、-20、-10、30、40、50mv有显著差异,30、40、50mv时明显升高。与对照组相比,TNF-α组APD则缩短APDso缩短43.6%,APD90缩短40%,APD50-APD90缩短37.9%有显著差异。结论：TNF-α可改变HL-1细胞的CACNA1c、KCNJ2、KCNH2、GJA1、 SLC8A1的mRNA表达,减少CAV1.2和ERG蛋白表达,减少L型钙通道离子电流、增加快速激活延迟整流钾电流电流和缩短APD, TNF-α对心房肌细胞的电重构中有重要作用。第二部分Jak-Stat信号通路对ERG的作用目的：Jak-Stat信号通路是细胞因子刺激的信号转导通路,参与细胞的增殖、分化、凋亡以及免疫调节等许多重要的生物学过程,在心脏发育、心肌肥厚和心肌纤维化发挥重要作用。TNF-α通过TNFR2激活Jak-Stat信号通路,在房颤电重构的作用尚不清楚。本部分研究初步探讨TNF-α刺激HL-1心房肌细胞后,Jak-Stat信号通路会对其电重构是否产生影响。方法：不同浓度TNF-α刺激HL-1细胞24小时后,用蛋白印迹法检测JAK1、 STAT3表达,siRNA敲除STAT3,用蛋白印迹法检测STAT3和ERG水平,同时用膜片钳方法观察对照组和实验组(siRNA-STAT3) IKR电流和APD。所有数据用均数±标准差(Mean±SD)表示,Ikr、APD采用两独立样本t检验。用SPSS13.0统计软件分析,以P<0.05为有统计学意义。结果：蛋白印迹法发现TNF-α0ng/ml、25ng/ml、50ng/ml,慢性刺激24小时后,JAK1和STAT3表达增多。予siRNA48小时,加入TNF-a24小时后,siRNA-negative组的STAT3和ERG明显高于siRNA-STAT3-1组、3iRNA-STAT3-2组、siRNA-STAT3-3组、siRNA-STAT3-pool组,STAT3和ERG表达减少。膜片钳观察发现与对照组相比,siRNA组快速激活延迟整流钾电流平台期和尾电流的电流密度减少,平台期电流密度在指令电位-30、30、40、50mv有显著差异,30、40、50mv时明显减低,尾电流电流密度在指令电位-30、-20、-10、10、20、30、40、50mv明显减低,有显著差异。与对照组相比,siRNA组APDso延长69%,APD90延长60%,APD50-APD90延长57.1%。我们通过t检验,发现siRNA组平台期电流密度、尾电流电流密度与正常细胞的相比明显下降,APD50、APD90仍短于正常细胞,但无统计学差异。结论：TNF-α可使HL-1细胞JAK1和STAT3表达增加,而抑制siRNA后明显减少ERG表达和降低快速激活延迟整流钾电流平台期和尾电流的电流密度,并低于无干预的HL-1细胞,同样能使APD50和APD90接近与无干预的HL-1细胞,说明STAT3对ERG是有影响的,并在TNF-α对HL-1细胞电重构的过程中起到作用,提示将来可能作为房颤治疗的一个靶点。更多还原

【Abstract】 With the aging of the population and the average life expectancy, the increasing incidence of atrial fibrillation (atrial fibrillation, AF), and the population mortality, morbidity is very significant. Is expected that China’s population will reach1.45billion in2020, accounting for about20%of the total population60years of age or older, health-care workers faced with the daunting atrial fibrillation prevention and treatment. Atrial fibrillation research in recent years, the rapid progress of drug therapy and radiofrequency catheter ablation has made some progress, but many poor patient outcomes, it is necessary to further study its pathogenesis, seeking new therapeutic approaches.Atrial fibrillation initiating many factors, with the prolonged duration of AF, atrial electrophysiology and cell characteristics changed sustained atrial fibrillation, a process called electrical remodeling, the main features of the action potential duration (APD) and atrialshorten the effective refractory period (ERP) and the increase in dispersion, reduced rate adaptive atrial conduction velocity, wavelength of the atrial contraction atrial fibrillation easily induced and increased stability. While the atrial refractory period and action potential shortening, net decrease of flow inward ion (Ca2+or Na+), net increase in outward (K+) ion flow, the gap junctions decrease closely relatedAtrial electrical remodeling occurred basis of ion channels damage or functional changes, the mechanism of complex, early studies suggest that atrial natriuretic peptide, atrial muscle ischemia with ATP-sensitive potassium channel, autonomic nervous system, calcium overload and metabolic disorders may be roomflutter electrical remodeling contacts in recent years focused on the pathway of oxidative stress and damage. Number of studies have confirmed that patients with AF right atrial appendage tissue, plasma high-sensitivity C-reactive protein (hs-CRP), tumor necrosis factor (TNF-alpha), interleukin-6(IL-6) compared with sinus rhythm group was significantly higher, TNF-alpha serum concentrations in paroxysmal, persistent and permanent atrial fibrillation was progressive increment that oxidative stress and inflammation can directly lead to atrial fibrillation pathophysiological changes. The animal was found that TNF-alpha can affect the the rat ventricular L-type calcium current, CX40, the role of other ion channels and gap link is not clear.Inflammatory cytokines TNF-alpha, IL-6, IL-4, involved in the activation and regulation of the JAK-STAT signaling pathway, JAK-STAT signaling pathway can upregulate cyclooxygenase (COX-2), nitric oxide synthase (NOS2), vascularendothelial growth factor (VEGF), antioxidant manganese superoxide dismutase (MnSOD), metallothionein (MT1and MT2), matrix metalloproteinase, thus affecting cardiac hypertrophy and cardiac remodeling. The study found that resveratrol by blocking JAK/STAT1passage so as to achieve the control of interferon-mediated macrophage migration can reduce the inflammatory response of vascular damage, and reduces the formation of arterial thrombus and plaque. STAT3gene knockout mice found increased interstitial fibrosis in the heart, increased TNF-alpha secretion, dilated cardiomyopathy and impaired cardiac function; knockout of STAT3gene or JAK2inhibitor AG490reduced expression of STAT3, myocardial infarct size can increase; Adawi study found that in the rat model of myocardial infarction, to the AG490Kv4.3mRNA expression shows that the JAK-STAT signaling pathway is involved in the pathophysiology of cardiovascular disease.Examined the effects of TNF-alpha chronic stimulation HL-1cells, to explore the effects of TNF-alpha on the part of the ion channel and gap connected JAK-STAT signaling pathway involved in the regulation of inflammatory cytokines TNF-alpha under the mechanisms of electrical remodeling. This experiment is divided into two parts.Part Ⅰ TNF-alpha on the HL-1atrial myocytes electrical remodelingObject:TNF-alpha involved in coronary heart disease, heart failure, rheumatic heart disease, congenital heart disease and a variety of cardiovascular disease pathological processes in the pathogenesis of atrial fibrillation is unclear. This part of the experiment in HL-1cells after stimulation by different concentrations of TNF-alpha, by detecting electrical remodeling related genes, to explore the effects of TNF-alpha on the HL-1cells electrical remodeling.Methods:TNF-alpha25ng/ml,50ng/ml stimulate HL-1cells after24hours, total RNA was extracted and total protein, using real-time polymerase chain reaction (RT-PCR) to detect CACNA1C, KCNJ2, KCNH2, KCNQ1, GJA5GJA1SLC8A1mRNA expression was measured by Western blot (Western bloting) Cav1.2and ERG expression, and the control group and the experimental group Ica, L, IKR current and APD recorded with the patch-clamp method. PCLAMP10.2software on a single whole-cell recording data and graphics conversion, Prism3.0software for data curve fitting and draw ion channel current density and APD, current density (pA/pF)=current intensity/capacitor.Results:RT-PCR, found1.CACNA1c mRNA levels decreased with the increase of TNF-alpha concentration, control group and the experimental group had significant difference (p<0.01), while the experimental group TNF-a50ng/ml group was significantly lower than25ng/ml (p<0.01).2.KCNJ2mRNA levels increase with the increase of TNF-alpha concentration, but control group and25ng/ml were no significant differences have a significant difference (p<0.05) with50ng/ml was no significant difference between the experimental group.3.KCNH2mRNA experimental group significantly higher than those in the control group (p<0.05), but no significant difference between the experimental groups. No significant difference between the the4.KCNQ1mRNA experimental and control groups. No significant difference between the the5.GJA5mRNA experimental and control groups.6.GJA1mRNA levels increase with the increase of TNF-alpha concentration, but the control group and25ng/ml group no significant difference between the control group,25ng/ml,50ng/ml significant difference (p<0.05).7.SLC8A1mRNA levels increase with the increase of TNF-alpha concentration, there is a significant difference the control group with the experimental group25ng/ml and50ng ng/ml(p<0.05and p <0.01), and25ng/ml group between50ng/ml group there are significant differences.Western blot found calcium channel Cavl.2decrease with the increase in TNF-alpha concentration and potassium ion channel ERG With the increase in TNF-alpha increase.By the the patch clamp detected with the control group compared to the decrease in TNF-alpha group L-type calcium ion current density, current density in the command potential-10mv,0mv,10mv,20mv,30mv,40mv decreased there were significant differences. Compared with the control group, TNF-alpha group quickly activate the delayed rectifier potassium current plateau and the tail current increase in current density, the current density of the plateau in the command potential-40,-30,-20,-10,30,40,50mv there are significant differences,30,40,50mV significantly increased. Compared with the control group, TNF-alpha group APD shortened APD50shortening of43.6%, APD90shortened to40%, the APD50-APD90shortening37.9%were significantly different.Conclusion:TNF-alpha can change the HL-1cells CACNA1C of KCNJ2, KCNH2, GJA1,, SLC8A1mRNA expression to reduce Cav1.2and ERG of protein to reduce the L-type calcium ion current to increase rapidly activating delayed rectifier potassium current currentand shorten the APD, TNF-alpha involved in electrical remodeling of atrial myocytes and in which there is an important role.Part Ⅱ Role of Jak-Stat signal pathway on ERGObject:JAK-Stat signaling pathway is cytokine-stimulated signal transduction pathways involved in cell proliferation, differentiation, apoptosis and immune regulation and many other important biological processes, play an important role in the development of the heart, myocardial hypertrophy and myocardial fibrosis. TNF-alpha TNFR2activates the JAK-Stat signaling pathway in atrial fibrillation electrical remodeling is unclear. This part of the study was to explore TNF-alpha stimulation HL-1atrial myocytes after the JAK-Stat signaling pathway of its electrical remodeling whether or not to have an impact.Methods:different concentrations of TNF-alpha stimulation of HL-1cells for24hours, with the western blot assay JAK1, STAT3expression, siRNA knock STAT3, was detected using Western blot STAT3and ERG level was observed in the control group, while using the patch-clamp methodand the experimental group (siRNA-STAT3) IKR current and APD.Results:Western blot analysis found that TNF-a Ong/ml,25ng/ml,50ng/ml, the chronic irritation after24hours, JAK1and STAT3expression increased. To siRNA48hour, TNF-a24hours after the STAT3and ERG of siRNA-negative group was significantly higher than that siRNA-STAT3-1group, siRNA-STAT3-2group, siRNA-STAT3-3group, siRNA-STAT3-pool group, The STAT3and ERG decreased expression.Patch-clamp observed compared with the control group, siRNA group rapid activation of the delayed rectifier potassium current platform and the current density of the tail current decreases, the current density of the plateau in the command potential significant difference-30,30,40,50mv,30,40,50mV significantly reduce the current density of the tail current at the command potential-30,-20,-10,10,20,30,40,50mV was significantly reduced, there are significant differences. Compared with the control group, siRNA group APD50extend the69%APD90extend the60%, the APD50-APD90extended to57.1%. Statistical methods, we found the siRNA group plateau current density, compared to the the tail current current density and the normal cells decreased significantly, APD50, APD90still shorter than normal cells, but no significant difference.Conclusion:TNF-alpha HL-1cells can JKA1and STAT3expression increased, while inhibiting siRNA to significantly reduce ERG expression and reduce the HL-rapid delayed rectifier potassium current platform and the current density of the tail current, and less than no interventionl cells, the same make the APD50and APD90close to the intervention HL-1cells. Description the STAT3ERG impact, and play a role in the process of reconstruction of TNF-alpha on the HL-1cells power, suggesting that futuremay be a target for the treatment of atrial fibrillation.更多还原