【作者】 宋忠举；

【导师】 闫素华；

【作者基本信息】 山东大学 ， 内科学， 2008， 硕士

【摘要】 研究背景及目的卫生部2006年调查资料显示:我国糖尿病患病率约为3%,患病人数高达2300万人,而血糖调节异常的人群则为3300万人。作为最常见的慢性病之一,糖尿病给我国人口健康及社会发展带来巨大负担。糖尿病心脏自主神经病变(diabeticcardiac autonomic neuropathy,DCAN)是糖尿病最常见的并发症之一,由于诊断标准的不同,其发生率在1%～90%。DCAN严重影响糖尿病患者的预后:T(?)yry等研究发现,在伴自主神经病变临床表现的2型糖尿病患者中,5年死亡率达50%,其中心源性猝死(sudden cardiac death,SCD)占28%;Sivieri等也报道1型糖尿病患者自主神经病变会导致QT间期延长,进一步引起致命性心律失常而发生SCD。而临床发现绝大多数SCD发生的基础机制是心室颤动(ventricularfibrillation,Vf),对猝死前的心电图分析发现心室颤动占75%～90%,故室性心律失常(ventricular arrhythmias,VAs)的发生对DCAN患者的预后有重大意义。另有研究提示糖尿病患者的心脏自主神经病变是导致其室性心律失常发生的原因之一,且室性心律失常发生的严重程度也可以反映DCAN的严重程度。但糖尿病对心脏自主神经的影响及该影响与室性心律失常的关系尚需进一步研究阐明。本研究旨在:1)建立兔糖尿病模型,采用免疫组织化学及实时荧光定量RT-PCR,研究糖尿病对心脏自主神经(交感神经)的影响;2)采用程序电刺激(programmed electrical stimulation,PES)的方法诱发室性心律失常,探讨糖尿病心脏自主神经(交感神经)的变化对室性心律失常易感性的影响及两者之间的关系,最终为DCAN患者室性心律失常的防治提供理论依据。材料与方法26只雄性新西兰大白兔测空腹血糖(fasting blood sugar,FBS)后随机分为2组:1)糖尿病组兔(diabetes mellitus group,DM组,n=16)制作糖尿病模型:10%的猪油、37%的蔗糖和53%的基础饲料混合制成的高脂高糖饲料饲养2月后由耳缘静脉注射四氧嘧啶(80mg/kg体重),以注射后72h和7d时两次FBS均≥14mmol/L作为糖尿病成模的标准;如未达标,则重复注射四氧嘧啶一次,仍未达标者则剔除;成模后改为普通饲料继续喂养6个月。2)对照组兔(control group,C组,n=10)一直用普通饲料同期喂养,糖尿病组造模时以等量生理盐水耳缘静脉注射。共喂养8个月后,所有成活的新西兰兔进行程序电刺激诱发室性心律失常实验,用免疫组化及实时荧光定量逆转录聚合酶链反应(RT-PCR)方法检测酪氨酸羟化酶(tyrosine hydroxylase,TH),以其作为交感神经标记物,观察两组实验兔左室近、中、远段及右室中段、室间隔中段等部位交感神经的分布密度和TH mRNA基因表达水平的变化。结果DM组实验兔有3只未成模,2只因严重高血糖死亡,最终有11只兔达到成模标准并存活至实验结束。C组实验兔均存活至实验结束。程序电刺激诱发室性心律失常实验发现,20%(2/10)的C组实验兔诱发出心室颤动(Vf);在DM组,则有27.3%(3/11)的实验兔诱发出室性心动过速(ventricular tachycardia,VT),9。1%(1/11)的实验兔诱发出心室扑动(ventricular flutter,VF),27.3%(3/11)的实验兔诱发出心室颤动(Vf),总室性心律失常诱发率达63.6%(7/11)。两组间室性心律失常诱发率存在统计学差异(P＜0.05)。免疫组织化学研究发现C组TH阳性神经纤维主要分布在血管周围,与血管相伴行,心肌束间的结缔组织也有分布;左室近段多见粗大神经纤维主干,左室远段多为纤细神经纤维末梢,中段则以神经纤维末梢为主,仍可以见到稍细的神经纤维干:神经纤维密度在左室近、中段较高,左室远段较低,但各部位之间无统计学差异(P＞0.05)。DM组TH阳性神经纤维的分布与C组相似,但神经纤维密度在左室近、中、远段及右室中段、室间隔中段等部位均明显低于C组相对应部位(P＜0.05,其中左室近段之间P=0.035,余部位之间P＜0.01)。DM组左室远段神经纤维明显减少,其神经纤维密度明显低于左室近、中段(均P＜0.01),左室中段神经纤维密度明显低于左室近段(P＜0.01);左室中段神经纤维密度低于右室中段(P＜0.05),而左室中段、右室中段神经纤维密度与室间隔中段相比均无统计学差异(均P＞0.05)。实时荧光定量RT-PCR方法检测两组各部位TH mRNA表达水平发现C组各部位间TH mRNA表达水平无统计学差异(P＞0.05);DM组各部位TH mRNA表达水平较C组各相应部位也均下降(P＜0.05,其中左室近段之间P=0.043,右室中段之间P=0.019,余部位之间P＜0.01);DM组左室远段TH mRNA表达水平较左室近、中段明显下降(均P＜0.01),左室中段TH mRNA表达水平较左室近段也明显下降(P＜0.01);左室中段TH mRNA表达水平较右室中段下降(P=0.04),而左室中段、右室中段TH mRNA表达水平分别与室间隔中段相比也均无统计学差异(均P＞0.05)。两种检测方法所得结果大体一致。结论1糖尿病对兔心脏交感神经有明显的损害,表现为酪氨酸羟化酶阳性神经纤维密度和TH mRNA表达水平的降低,且各部位降低的程度有所不同:左室由近段至远段降低的程度逐步加重,左室中段降低的程度重于右室中段,表明左室近、中、远段之间以及左室与右室之间出现了交感神经支配的失衡。2糖尿病对兔心脏交感神经的影响增加了室性心律失常的易感性。更多还原

【Abstract】 Background and objectsAccording to the investigation documents of the Department of Health in 2006, the morbidity rate of diabetes of our country is about 3%, and the diabetic populations are 23 million, but the populations of impaired blood sugar modulation are 33 billion. Diabetes, as one of the most popular chronic diseases, brings huge burdens on the population health and society development of our country. Diabetic cardiac autonomic neuropathy (DCAN) is one of the common complications of diabetes. The prevalence rates of DCAN are 1% to 90% because of the differences in the diagnostic criteria. DCAN aggravates the prognosis of the diabetic badly. Toyry et al reported that the mortality of the type 2 diabetics with autonomic neuropathy clinical manifestation in 5 years is 50%, and 28% of them died of sudden cardiac deaths (SCDs). Sivieri et al also reported that DCAN could prolong the QT intervals to induce fatal arrhythmias, which might lead to SCDs in the type 1 diabetics. In clinical cases, the basal mechanism of most SCDs is ventricular fibrillation (Vf), and the analyses of electrocardiograms before sudden deaths reveal that 75% to 90% of sudden deaths are induced by ventricular fibrillation, so the incidence rate of ventricular arrhythmias (VAs) in diabetics may reflect their prognoses. Additional researches prompted that DCAN was one of the reasons to induce ventricular arrhythmias in the diabetics and the severity of ventricular arrhythmias could also reflect the severity of DCAN. However, the influences of diabetes on the cardiac sympathetic nerve and the relationships between the influences and ventricular arrhythmias are still needed to be researched and illuminated.The main objects of this study are: 1) Diabetic models were erected in rabbits, and immunohistochemistry and real time fluorescent quantitation reverse transcription polymerase chain reaction (RT-PCR) were used to observe the influences of diabetes on the cardiac autonomic nerve (sympathetic nerve). 2) Programmed electrical stimulation (PES) was used to induce ventricular arrhythmias, and the relationships were approached between the changes of cardiac autonomic nerve (sympathetic nerve) in diabetes and the incidences of inducible ventricular arrhythmias. The final object is to provide theory foundations for the prevention and cure of ventricular arrhythmias induced by DCAN.MethodsTwenty-six male New Zealand white rabbits were randomly assigned to two groups after fasting blood glucoses (FBS) were measured. Diabetes mellitus group rabbits (DM group, n-16) were induced into diabetic models. This group of animals was fed with the high-fat/high-sucrose diet in the first two months, which was made from 10% lard, 37% sucrose and 53% regular rabbit chow, and then every rabbit was injected with alloxan (80mg/kg body weight) via the ear vein. At 72 hours and 7 days after the injection, rabbits who demonstrated fasting blood glucose levels of 14mmol/L or above were accepted as diabetic rabbits. If the fasting blood glucose of rabbit was lower than 14mmol/L, another injection of alloxan was given. The rabbit which fasting blood glucose was still lower than 14mmol/L was rejected. Then the diabetic rabbits were fed with the regular rabbit chow for 6 months until the end of the experiment. The control group rabbits (C group, n=10) were always fed with the regular rabbit chow at the same time. They were injected with normal saline via the ear vein when the DM group rabbits were injected with alloxan. 8 months after the beginning, programmed electrical stimulation was applied to induce ventricular arrhythmias. Then immunohistochemistry and real time fluorescent quantitation RT-PCR were applied to detect tyrosine hydroxylase (TH) protein and gene expression, as the marker of sympathetic nerve, to reveal the changes of sympathetic nerve innervation densities and TH mRNA gene expression levels in different cardiac segments of the two groups of rabbits.Results3 of the DM group rabbits did not catch the standard of diabetes, 2 died of severe high blood glucoses, and 11 were accepted as diabetic rabbits and survived to the end of the experiment. The C group rabbits all survived to the end of the experiment.Ventricular arrhythmias induced by programmed electrical stimulation occurred in 2/10 (20%) rabbits in the C group, which are all ventricular fibrillation. In the DM group, 27.3% (3/11) of the rabbits had ventricular tachycardia (VT), 9.1% (1/11) had ventricular flutter (VF), 27.3% (3/11) had ventricular fibrillation, and the total ventricular arrhythmias incidence was 63.6% (7/11). There are statistical differences between the two groups (P<0.05).In the C group, the TH positive nerve fibers mainly distribute at the perivascular regions and go along with the vessels, some in the connective tissue between the myocardial bundles. A lot of thick nerve fiber stems can be seen in the proximal segments of left ventricles, and most nerve fibers are slender in the distal segments of left ventricles, while both slender nerve fibers and slightly slender nerve fiber stems coexist in the middle segments of left ventricles. The densities of TH positive nerve fibers were higher in the proximal and the middle segments of left ventricles, lower in the distal segments of left ventricles, but there are not statistical differences among all the five parts of the C group (P>0.05). The distribution of TH positive nerve fibers in the DM group are similar to that in the C group, but the densities of the nerve fibers in the proximal, middle and distal segments of left ventricles and in the middle segments of right ventricles and interventricular septa all decrease compared with the corresponding segments in the C group (P<0.05, P=0.035 between the proximal segments of left ventricles in the two groups, P<0.01 between all the other corresponding segments in the two groups). In the DM group, the nerve fibers densities decrease obviously in the distal segments of left ventricles, the nerve fibers densities in which are much lower than those in the proximal segments and the middle segments of left ventricles (P<0.01, respectively). The nerve fibers densities in the middle segments of left ventricles are much lower than those in the proximal segments of left ventricles, too (P<0.01). The nerve fibers densities in the middle segments of left ventricles are lower than those in the middle segments of right ventricles (P<0.05). But there are not statistical differences in the nerve fibers densities between the interventricular septa and the middle segments of left ventricles or right ventricles (P>0.05, respectively). There are not statistical differences in the expression levels of TH mRNA among all the five parts in the C group (P>0.05). The expression levels of TH mRNA in all the five parts in the DM group decrease compared with the corresponding segments in the C group, too (P<0.05, P=0.043 between the proximal segments of left ventricles in the two groups, P=0.019 between the middle segments of right ventricles in the two groups, P<0.01 between the other corresponding segments in the two groups). In the DM group, the expression levels of TH mRNA in the distal segments of left ventricles are much lower than those in the proximal segments and the middle segments of left ventricles (P<0.01, respectively). The expression levels of TH mRNA in the middle segments of left ventricles are also much lower than those in the proximal segments of left ventricles (P<0.01). The expression levels of TH mRNA in the middle segments of left ventricles are lower than those in the middle segments of right ventricles (P<0.05). But there are not statistical differences in the expression levels of TH mRNA between the interventricular septa and the middle segments of left ventricles or right ventricles (P >0.05, respectively). The expression levels of TH mRNA detected by real time fluorescent quantitation RT-PCR are coincident to the TH positive nerve fibers densities revealed through immunohistochemistry in every segment of the two groups.Conclusion1 Diabetes damages the cardiac sympathetic nerve of rabbits conspicuously. The damages are displayed by the decline of TH positive nerve fibers densities and expression levels of TH mRNA in the DM group compared with in the C group, and the decreasing degrees among the five cardiac segments in the DM group are different, which aggravate gradually from the proximal segments to the distal segments of left ventricles and are heavier in the middle segments of left ventricles than in the middle segments of right ventricles. There are disequilibrium of sympathetic innervation among the distal segments, the proximal segments and the middle segments of left ventricles and between the middle segments of left ventricles and right ventricles.2 The influence of diabetes on the cardiac sympathetic nerve of rabbits increases the affectabilities of ventricular arrhythmias.更多还原