【作者】 宋光远；

【导师】 杨跃进； 吴永健；

【作者基本信息】 中国协和医科大学 ， 内科学， 2009， 博士

【摘要】 目的:研究链脲佐菌素(streptozotocin,STZ)诱导的1型糖尿病(diabetes mellitus,DM)大鼠急性心肌梗死(acute myocardial infarction,AMI)后的存活率、心脏结构和功能以及心肌纤维化时间依赖性变化。方法:Sprague-Dawley(SD)大鼠应用STZ一次性腹腔注射(65mg/dl)诱导1型糖尿病10周,未进行降糖治疗,在诱导前以及诱导后3天、7天、30天和70天记录体重、检测血糖并应用心肌透射电镜扫描观察糖尿病大鼠心肌超微结构的变化;结扎冠状动脉前降支,不进行再灌注治疗,计算大鼠在结扎后1天、7天、14天、28天和56天的存活率;应用超声心动图和血流动力学监测糖尿病大鼠在AMI后上述5个时间点的心脏功能;应用Masson’s染色来定量评价其非梗死区心肌纤维化的程度。结果:诱导糖尿病10周后,糖尿病大鼠的血糖显著高于非糖尿病组大鼠(489±28vs.112±13mg/dl,P＜0.001);同时其体重从第3日起与非糖尿病大鼠有显著差别,并且差异进行性加大,至10周时其体重显著低于非糖尿病大鼠(244.8±23.6 vs.516.3±48.6克,P＜0.001)。心肌电镜扫描观察到糖尿病组大鼠心肌细胞的各带结构不清,走行紊乱,核周围线粒体增多,线粒体周围糖原和脂滴聚集,线粒体肿胀。部分心肌纤维出现纤维溶解坏死,同时伴凝固性坏死。AMI后糖尿病大鼠存活率明显低于非糖尿病大鼠,非糖尿病组在对应时间点的存活率为82.9%(58/70)、80.0%(48/60)、80.0%(40/50)、77.5%(31/40)和76.7%(23/30),而糖尿病组在1天、7天、14天、28天和56天的存活率分别为67.2%(44/64,P＜0.05)、64.8%(35/54,P=NS)、61.4%(27/44,P＜0.05)、52.9%(18/34,P＜0.05)和41.7%(10/24,P＜0.05)。超声心动图和血流动力学监测显示,糖尿病大鼠AMI前存在舒张功能不全;AMI后两组大鼠心功能均明显下降,糖尿病大鼠心功能恶化更加显著(EF 37.3±5.4%vs.48.8±4.1%,P＜0.01 and FS 17.0±1.4%vs.23.7±2.5%,P＜0.05);左心室扩张更加明显(增加的LVEDD 47.5±5.2%vs.26.8±4.9%,P＜0.001);心肌纤维化染色发现糖尿病大鼠AMI28天和56天时非梗死区纤维化相对于非糖尿病大鼠更加明显(10.3%±1.5%vs.3.8%±0.9%,P＜0.001 and 17.5%±2.1%vs.9.8%±1.2%,P＜0.001)。结论:一系列病理生理学改变显示,糖尿病大鼠AMI后易发心力衰竭的原因早期是由于严重的心脏舒张功能不全,晚期则是由于其心功能不全和心肌重塑加速所致。目的:研究糖尿病与非糖尿病大鼠急性心肌梗死后基因表达的差异。方法:SD大鼠应用STZ一次性腹腔注射(65mg/dl)诱导1型糖尿病10周,未进行降糖治疗;结扎冠状动脉前降支,应用大鼠全基因序列的基因芯片、Real time-PCR来检测AMI后1天、7天、14天、28天和56天糖尿病大鼠左心室非梗死区基因表达的变化。结果:AMI后,糖尿病大鼠与非糖尿病大鼠相比生存率明显降低,如前所述,病理生理学变化表明DM能够加速AMI后心肌重塑的进展。基因芯片分析显示随着时间的变化,两组大鼠基因表达显著不同,糖尿病大鼠AMI后左心室非梗死区心肌组织有1221个基因表达与非糖尿病大鼠显著不同,其中770个表达上调,451个表达下调;对差异基因进行功能分析发现其涵盖糖代谢、脂肪酸代谢、细胞外基质、凋亡、氧化还原和磷酸酶活性等诸多方面。无监督聚类分析发现糖尿病大鼠AMI后左心室非梗死区1-7天的基因表达变化和非糖尿病大鼠相似,14天时的基因表达变化与非糖尿病大鼠14和28天相似,而其28和56天的基因变化和非糖尿病大鼠56天相似,显示糖尿病大鼠AMI后重塑相关基因的表达谱提前1-2周。根据筛选条件,应用到无监督聚类分析的基因为164个,其中118个已经被命名,大部分被证实与心脏重构密切相关,例如富含亮氨酸的PPR模体(白介素-6信号通路)、原骨胶原Ⅰ型和Ⅲ型、纤维连接蛋白1、RT1,and TIMP-1等等。结论:STZ诱导的1型糖尿病大鼠急性心肌梗死后心功能的恶化可能与相关基因表达谱的前移和过量表达有关。目的:观察糖尿病大鼠AMI后梗死周边区的心肌细胞凋亡的时间依赖性变化。方法:SD大鼠应用STZ一次性腹腔注射(65mg/dl)诱导1型糖尿病10周,未进行降糖治疗;结扎冠状动脉前降支,取材,应用免疫细胞化学Tunel法检测细胞凋亡。取材左心室梗死周边区组织(距离心肌梗死边缘2.5mm范围),应用免疫印迹(Western blot)法半定量分析凋亡相关蛋白Bax和Bcl-2蛋白水平的含量;应用实时定量PCR(real time-PCR)法定量分析结缔组织生长因子(CTGF)和caspase-3mRNA水平的表达。结果:研究显示STZ诱导10周后,糖尿病大鼠心肌组织凋亡指数显著高于非糖尿病大鼠(3.2±0.5 vs.1.4±0.3,P＜0.01);在急性心肌梗死后的1天(20.1±1.8 vs.16.9±1.5,P＜0.05)、7天(16.1±1.7 vs.12.9±1.3,P＜0.01)、14天(16.1±1.7 vs.13.1±1.4,P＜0.05)、28天(15.2±1.8 vs.11.9±1.5,P＜0.01]和56天(16.3±1.6 vs.9.6±1.2,P＜0.001)时其左心室梗死周边区心肌细胞进行性凋亡,凋亡指数(括号中数值)显著高于非糖尿病大鼠。和非糖尿病组大鼠相比,Western blot法半定量分析显示糖尿病大鼠急性心肌梗死后1天、7天、28天和56天促凋亡蛋白Bax水平显著增加(P＜0.001);而抑凋亡蛋白Bcl-2水平在AMI后1天、14天、28天和56天显著降低(P＜0.001)。实时定量PCR结果显示CTGF和caspase-3mRNA的水平较非糖尿病大鼠显著增加。结论:与非糖尿病大鼠相比,糖尿病大鼠急性心肌梗死后左心室梗死周边区的细胞进行性凋亡可能会导致其左心室扩大和心肌纤维化的程度显著较高,从而更加容易发生心力衰竭;其Bax和Bcl-2的蛋白水平提示其细胞凋亡通路可能包含线粒体凋亡通路。更多还原

【Abstract】 Objective:In spite of numerous studies in diabetic animal models,to our knowledge,myocardial performance,especially time course study was never performed in untreated diabetic myocardial infarction(MI) rat model without reperfusion.Methods:In the present study,myocardial function and structure changes of myocardium were examined in untreated streptozotocin(STZ)-induced diabetic MI rat model without perfusion. DM was induced with a single intraperitoneal injection of STZ(65 mg/kg),and body weight was recorded and serum glucose levels were determined at baseline,day 3,day 7,day 30 and day 70 after STZ injection by using One Touch Sure Step test strips and meter(Life Scan,Johnson & Johnson,Warren NJ,USA).Ten weeks after DM induction without any therapy,the left anterior descending coronary artery of diabetic Sprague-Dawley rats was ligated to induce no-reperfusion MI model Tissue structure of myocardium,two-dimensional echocardiography,and hemodynamic studies were utilized to monitor the time course changes of above relating parameters up to 56 days.Results:Ten weeks after DM established,serum glucose levels were significantly higher in the diabetic rats(489±28mg/dl) than in the non-diabetic animals(112±13mg/dl,P＜0.001).Body weights were significantly lower in the diabetic rats(244.8±23.6g) than that of non-diabetic ones (516.3±48.6g,P＜0.001).TEM examination showed that the heart of diabetic rats contained increased lipid droplets and glycogen particles around mitochondria and scattered mitochondrial damage(swelling and disrupted cristae);in addition,cardiac muscle fibers were identified to be crumbly in the majority of the diabetic rats,and the basal lamina of regional small vessels was thicker in some regions.After AMI,Diabetic rats experienced lower survival rate in both acute phase(1,7 and 14 days) and chronic phase(28 and 56 days) compared with non-diabetic rats at corresponding time points.During 56 days after myocardial infarction,worse left ventricular function(EF 37.3±5.4 vs.48.8±4.1,P＜0.01 and FS 17.0±1.4 vs.23.7±2.5,P＜0.05) and larger the left ventricular diameter(increased LVEDD 47.5±5.2%vs.26.8±4.9%,P＜0.001) were identified in diabetic rats than in non-diabetics.Furthermore,diabetic rats showed more serious myocardial fibrosis by quantification of fibrosis area in the remote zone of left ventricular free wall compared with non-diabetic ones at 28 and 56 days after MI(Masson’s quantitation 10.3%±1.5%vs.3.8%±0.9%,P＜0.001 and 17.5%±2.1%vs.9.8%±1.2%,P＜0.001).Conclusion:The different patterns of the pathophysiologic changes in the untreated STZ-induced diabetic MI rat model without reperfusion might suggest that in the early phase after AMI,LV diastolic dysfunction may account for the higher incidence of HF and death,while in the prolonged phase,both LV dysfunction and accelerated cardiac remodeling can be the motivation. Objective:To study the time-dependent effects of genetic changes in untreated Streptozotocin (STZ)-induced diabetic rat with MI.Methods:The left anterior descending coronary arteries were ligated 10 weeks after DM induction without any therapy and reperfusion.Microarray analysis and real time-PCR were utilized to monitor the time-dependent genetic changes in the remote zone of LV up to 56 days.Results:The gene expression related to cardiac remodeling in the remote zone was quite different as time elapses between both groups.Total 1221 genes were found to be differentially expressed between diabetics and non-diabetics,770 unregulated and 451 downregulated, involving in glucose metabolism,fatty acid metabolism,extracellular matrix,apoptosis, oxido-reductase activity,phosphoprotein phosphatase activity,etc.The gene expression at 1 and 7 days post acute MI in diabetic and non-diabetic group was similar,while such changes at day 14 in diabetic rats were comparable to both 14 and 28 days in non-diabetic rats;moreover,the pattern of gene expression at day 28 and 56 in diabetic rats was similar to the ones at day 56 in non-diabetic rats,which may indicate that upregulation of the genes was found one-two weeks earlier in diabetic rats than in non-diabetic rats.In primary examination,164 genes(118 named) were found to be candidates for hierarchical analysis,such as leucine-rich PPR-motif containing (interleukin-6 signaling pathway),procollagen typeⅠandⅢ,fibronectin-1,RT1,and TIMP-1,etc.Conclusion:The present data support that diabetes upregulates remodeling-related gene,which may be responsible for the worse outcomes in diabetes than in non-diabetes after myocardial infarction. Objective:To examine whether diabetes is associated with enhanced cardiomyocyte apoptosis and thus interferes with the post-infarction remodelling process in myocardium in rat.Methods:Ten weeks after intravenous streptozotocin(diabetic groups) or citrate buffer(controls) injection,myocardial infarction was produced by ligation of left descending coronary artery.Level of cardiomyocyte apoptosis was quantified by TUNEL method;Bax and Bcl-2 proteins were determined by western blot method;CTGF and caspase-3 were quantitated by real time-PCR.Results:The number of apoptotic cardiomyocytes was higher in diabetic than in non-diabetic rats after 10 weeks of DM induction(apototic index,3.2±0.5 vs.1.4±0.3,P<0.01).At 1d(20.1±1.8 vs.16.9±1.5,P<0.05),7d(16.1±1.7 vs.12.9±1.3,P<0.01),14d(16.1±1.7 vs.13.1±1.4,P<0.05), 28d(15.2±1.8 vs.11.9±1.5,P<0.01) and 56d(16.3±1.6 vs.9.6±1.2,P<0.001) after myocardial infarction,the apoptotic indices in the border zone of infarction area was higher in the diabetic as compared to non-diabetic rats.Correspondingly,Bax,connective tissue growth factor and caspase-3 expression were increased in diabetic compared with non-diabetic rats with myocardial infarction,whereas Bcl-2 expression was decreased.Conclusion:Apoptotic myocyte loss could be an important mechanism contributing to progressive dilatation of the heart and poor prognosis after myocardial infarction in diabetes.更多还原