【作者】 郭红阳；

【导师】 王玉堂；

【作者基本信息】 中国人民解放军军医进修学院 ， 内科学， 2008， 博士

【摘要】 目的应激可导致包括VT、Vf在内的SIA,同时伴有SCD增多。研究表明ANS、心肌缺血等因素导致CES降低是SIA发生的重要原因。同时HRV、TWA、HRT、细胞内钙浓度变化、CNS、遗传等因素对SIA的发生均产生重要影响。我们认为在上述机制中,有些可能是应激时ANS功能改变后的下游反应,而另一些则可能存在于ANS功能调控之外,这些机制之间的关系尚待进一步澄清。另外,尽管有证据提示ANS在SIA的发生中处于关键地位,但其中仍有许多具体的环节未明。本研究通过①分析应激状态下ANS功能变化及其与SIA的相关性,②分析应激状态下,交感神经信号的细胞传导的途径,以期阐明SIA发生的自主神经机制。方法健康雄性山羊15只,体重18-26kg。分为对照组、应激组和应激+倍他乐克干预组,每组5只山羊。采用声、光复合刺激方法建立急性应激模型。通过24小时Holter记录,分析心律失常的发生情况、HRV、TWA等指标。随后进行开胸实验,首先分离双侧颈交感神经,同步记录20分钟的电活动,并进行频谱和功率谱分析。之后于左侧第4肋间开胸,暴露心脏。应用自制的心外膜电极于心脏表面不同区域进行ERP检测,并观察此过程中心律失常和AD的出现情况。上述指标测量完毕后,主动脉放血除死动物,快速留取LA、LV全层肌组织,制备心肌细胞膜样本。应用放射性配基结合实验检测心肌β-AR密度,间接法检测AC活性,放射免疫分析法检测心肌cAMP水平。结果1.ST段分析Holter记录中ST分析显示,山羊应激过程中ST段均在正常范围之内。2.Holter记录时心律失常分析应激组和干预组的SVPB、SVT和VPB数量增多,且应激组多于干预组。SVPB(28.5±5.1个/h vs 102.6±10.9个/h vs 59.4±8.7个/h,P均<0.01);SVT(10.6±3.1阵/24h Vs 32.4±6.1阵/24h vs 18.8±4.2阵/24h,P<0.01或0.05);VPB(0.7±0.2个/hvs 10.5±1.8个/h vs 4.6±1.2个/h,P均<0.01);三组动物均未出现Af、vT、vF和Vf。3.HRV分析应激组和干预组SDANN和rMSSD均显著下降,但干预组高于应激组(SDANN:181.4±20.1ms vs 113.6±17.3ms vs 142.4±15.7ms,P＜0.01或0.05,rMSSD:45.8±5.7ms vs 24.6±5.5ms vs 37.2±4.2ms,P＜0.01或0.05)。无论白天还是夜间,应激组LFn及LF/HF均显著高于对照组和干预组(P＜0.0l或0.05),干预组高于对照组(P＜0.01或P＜0.05);应激组HFn与对照组和干预组相比显著降低(P＜0.01或0.05),且干预组低于对照组(P＜0.01);对照组白天与夜间的LFn、LF/HF和HFn有显著差异,前两者呈现出昼高夜低、而后者则为昼低夜高的变化模式(P＜0.05),应激组和干预组均无此特点。4.心律失常发生与HRV的相关分析三组动物SVPB和VPB与LF/HF比值间存在正相关直线关系。复相关系数分别为0.924、0.877、0.886和0.774、0.91 3、0.885。回归方程分别为:Y=-100.3+349.7X、Y=-1225.0+805.2X、Y=-99.0+419.4X和Y=-10.6+14.7X、Y=-211.6+110.7x、Y=-33.4+51.6x。5.TWA分析应激组和干预组TWA显著增加,但干预组TWA小于应激组(P＜0.01或0.05)。6.ERP及离散度分析应激组和干预组LA、LV的ERP较对照组显著缩短,干预组ERP较应激组有所延长。应激组和干预组ERP离散度显著增大,干预组离散度小于应激组(LA-ERP:134.3±5.2 vs 108.5±6.8 vs 119.1±5.9ms,D-LA—ERP:4.39±0.41 vs 5.77±0.48 vs 5.07±0.4 3ms,LV-ERP:208.5±6.1 vs 185.9±7.0 vs 197.9±6.7ms,D-LV-ERP:5.23±0.35 vs 6.45±0.40 vs 5.88±0.44ms,P＜0.01或0.05)。7.ERP测量过程中EAD的分析应激组EAD显著多于对照和干预组(P均＜0.01),后两组间无显著差别(2.2±1.1次vs 5.2±1.3次Vs 2.8±0.8次)。8.ERP测量时心律失常分析应激组有3只山羊、干预组有1只山羊诱发出了短阵Af,对照组未能诱发出Af,三组山羊Af诱发率无差别。应激组VPB(2.8±0.8个)较对照组(0.6±0.5个)和干预组(1.0±0.7个)显著增多(P＜0.01),但对照组和干预组间无差别。9.颈部交感神经放电活动分析双侧交感神经放电波形呈阵发簇状,在每一簇中,波峰数不同,持续时间不同,幅值在-50至+50μV,两侧神经放电活动并不完全同步。频谱和功率谱分析显示,与对照组相比,应激和干预组颈交感神经在51Hz、54Hz、148Hz处的放电峰值,以及在51-250Hz、51-140Hz,140-160Hz,160-250Hz频带内放电功率均显著增高(P＜0.05或0.01),但应激和干预组间无显著差别。对照组双侧颈交感神经放电活动无显著差异,而应激组和干预组双侧颈交感神经放电峰值和功率均不同(P＜0.05或0.01)。10.心肌β-AR密度与对照组心房肌和心室肌β-AR密度(80.4±12.1和50.2±11.3fmol/mg·protein)相比,应激组(110.5±17.4和78.2±16.5fmol/mg·protein)和干预组(103.8±15.8和72.4±15.2 fmol/mg·protein)显著增高(P＜0.01或0.05),但应激组和干预组间无显著性差异;三组动物K_D比较亦无差别。11.AC活性应激组心房肌和心室肌AC水平(67.2±12.6和58.7±10.2 pmol/g·s)高于对照组(34.7±7.1和23.3±4.3 pmol/g·s)和干预组(53.4±9.3和38.6±8.9 pmol/g·s)(P＜0.01或0.05),且干预组高于对照纽(P＜0.05)。12.cAMP含量与对照纽心房、心室肌cAMP含量(511.4±72.4和400.2±80.5pmol/g)相比,应激组(787.9±112.6和686.8±108.8pmol/g)和干预组(645.8±86.6和548.9±78.9pmol/g)显著增高(P＜0.01或0.05),干预组低于应激组(P＜0.05)。结论ANS在SIA的发生中起到了重要作用。应激山羊存在交感神经兴奋、迷走神经抑制,以及自主神经活动生理节律的丧失,从而诱发心肌ERP缩短、ERP离散度和TWA明显增大,最终导致SIA的显著增多。而两侧交感神经对心脏驱动状态的不同,可能是造成心肌ERP和复极离散度增加的重要原因。干预组心律失常较应激组减少,而且HRV、TWA、ERP离散度等指标部分改善,提示倍他乐克可能通过改善应激时ANS功能异常,提高心肌电稳态,从而减少SIA的发生。但倍他乐克未能改变应激源对机体的整体刺激,未能减少整体交感神经的激活和逆转两侧交感神经驱动状态的失衡,未能恢复HRV的生理节律,这些机制可能是干预组山羊SIA仍显著多于对照组的原因所在。β-AR—G蛋白—AC—cAMP途径是急性应激信号由胞外向胞内传递的重要通路,但仍需寻找其他可能的细胞信号传导机制。更多还原

【Abstract】 Objective:Stress can leads to SIA including VT、Vf,and increased SCD. Some studies indicated that myocardial electrical unstability indued by changed ANS function or myocardial ischemia are impotant inciting factors of the genesis of SIA.Meanwhile,the change of HRV、TWA、HRT、intracellular calcium concentration、CNS and hereditary factors play important roles in inducing SIA. But some of these factors are downstream reactions of ANS function changes, and some of them may be beyond the mechanism of ANS regulation,the relationship of them needs to be clarified.On the other hand,although there are some evidence showed that ANS is critical for inducing SIA,but many details are still unknown.This Study established a goat stress model and analyzed the changes of ANS and its relation to SIA,and the pathway of sympathetic signals transmiting across the myocardial cell membrane,in order to elucidate the autonomic mechanism of SIA.Methods:15 adult male goats(body weight 18-26 kg)were divided into control,stress and metoprolol groups.Noise and flash light were used for 24 hours to establish the acute stress model.By 24 hours electrocardiogram monitoring,arrhythmia、HRV、TWA were recorded and analyzed.Subsequently, the animals were anesthetized and cervical sympathetic nerves were dissociated bilaterally,after recording the electrical activities of nerves for 20 minutes, open-thoracic surgery were performed,epicardial leads were used to measure ERP in different zone of left atrium and ventricle,in the same time,the induced arrhythmias and AD were recorded.Subsequently,the animals were killed by bloodletting through aorta,the myocardial tissue were kept for preparing the myocardial membrane sample.Myocardialβ-AR density、AC activity and cAMP level were measured by radioligand binding assay and radioimmunoassay. Results:1.ST segment analysis ST segment were within normal range during the sress state.2.Arrhythmias during Holter recording stress and metoprolol groups have more SVPB、SVT and VPB(P＜0.01 or 0.05),and there is no Af、VT、VF and Vf occurring.3.HRV analysis Both SDANN and rMSSD decreased significantly in stress and metoprolol groups,but these indices are higher in metoprolol group than stress group(P＜0.01 or 0.05).Stress group have higher LFn and LF/HF ratio and lower HFn than control and metoprolol groups, and metoprolol guoup have increased LFn、LF/HF ratio and decreased HFn than control group(P＜0.01 or 0.05).LFn、LF/HF value of control group has high-in-daytime and low-in-night model,HFn is on the contrary,but nerther stress nor metoprolol group have similar features.4.The relation between arrhythmias and HRV There is linear relationship between arrhythmia(SVPB、VPB)and LH/HF ratio,correlation coefficient are 0.924、0.877、0.886 and 0.774、0.913、0.885 respectively,regression equation are Y=-100.3+349.7X、Y=-1225.0+805.2X、Y=-99.0+419.4X and Y=-10.6+14.7X、Y=-211.6+110.7X、Y=-33.4+51.6X respectively.5.TWA analysis TWA increased in stress and metoprolol group,but the latter is lower than the former(P＜0.01 or 0.05).6.ERP and its dispersion stress and metoprolol group have shortened ERP and increased ERP dispersion,and stress group have more significant change (P＜0.01).7.EAD analysis stress group have more EAD than the control and metoprolol groups(P＜0.01),but there is no difference between the control and metoprolol group,8.Arrhythmias during ERP measurement Af was only induced in 3 goats of the stress group and 1 goat of metoprolol group,and the incidence of Af are not different.Stress group have more VPB than control and metoprolol group(P＜0.01),there is no difference between the control and metoprolol group. 9.Cervical sympathetic electrical activity analysis the peak value in 51Hz、54Hz、148Hz and the power of 51-250Hz、51-140Hz,140-160Hz,160-250Hz are similarly and significantly increased in stress and metoprolol group(P＜0.05 or 0.01).In control group,there is no remarkable differences of peak value and power between the sympathetic nerves on the two sides of neck,but in stress and metoprolol groups,the right sympathetic nerve has higher peak value and power (P＜0.05 or 0.01).10.Myocardialβ-AR density stress and metoprolol groups have increased receptor density than control group,but there is no significant difference between the control and the metoporolol group,and K_D value is not different either.11.AC activity and myocardial cAMP level stress group has higher AC activity and cAMP level than the control and metoprolol groups (P＜0.01 or 0.05),and these indices of metoprolol group also higher than that of control group(P＜0.05).Conclusion:ANS play an important role in the genesis of SIA.Stressed goats have increased sympathetic activity and decreased vagal tone,and lost the normal circadian rhythm,which may induced shortened ERP and increased ERP dispersion and TWA amplitude,and ultimately induced SIA.The different drive of sympathetic nerves bilaterally may be the most important cause leading to increased dispersion of ERP and repolarization.Metoprolol improve the abnomal autonomic function of heart in stressed goats,and subsequently improve the abnormality of HRV,TWA,ERP dispersion,and enhance the myocardial electrical stability,and decrease the genesis of SIA,But metoprolol cannot inhibit the whole stress reaction of the body,and cannot inhibit sympathetic activity and imbalance of bilateral sympathetic drive,and cannot recover the normal circadian rhythm of HRV,so,the goats of metoprolol group still have relatively high incidence of SIA.β-AR—G protein—AC—cAMP chain is the important but not the only pathway by which the stress signals are transmitted across the myocardial cell membrane.更多还原