兔心肌缺血性损伤的超微结构变化及凋亡检测

目的　探讨实验性急性心肌梗塞时不同损伤区域心肌超微结构的变化及心肌细胞是否凋亡。方法　结扎左冠状动脉的左室支建立心肌缺血模型 ,采用透射电镜、原位末端探针标记 ,对不同损伤区心肌细胞的损伤情况进行研究。结果　电镜观察显示中心缺血区心肌组织呈现缺血坏死征象 ,而梗塞交界区呈现明显的心肌细胞凋亡征象。TUNEL检测结果显示中心缺血区未见心肌凋亡细胞发生 ,而梗塞交界区有心肌凋亡细胞检出。结论　急性心梗时 ,中心缺血区和梗塞交界区损伤存在不同的发生机制     

兔心肌缺血再灌注损伤的超微结构变化及凋亡检测

目的　探讨实验性心肌缺血再灌注损伤时心肌超微结构变化及心肌细胞凋亡的发生。方法　选家兔 6只 ,阻断左冠状动脉的左室支建立心肌缺血模型 ,达到预定的缺血时间后 ,使血管再通 ,建立心肌的缺血再灌注模型。采用透射电镜、原位末端探针标记 (TUNEL)对不同损伤区心肌细胞的损伤情况进行研究。结果　电镜观察显示缺血再灌注损伤中心区呈现明显的心肌细胞凋亡征象 ,在缺血再灌注损伤交界区可见心肌细胞凋亡的早期征象 ,TUNEL检测结果显示缺血再灌注损伤中心区出现较多的凋亡阳性细胞 ,而损伤交界区出现较少的凋亡阳性细胞。结论　心肌细胞凋亡是心肌缺血再灌注损伤的重要特征 ,这与心肌的缺血性损伤存在不同     

兔实验性心肌缺血再灌注损伤与心肌细胞凋亡的关系

目的　探讨实验性心肌缺血再灌注损伤时心肌细胞是否凋亡及凋亡率的大小。方法　阻断左冠状动脉的左室支建立家兔心肌缺血模型 ,达到预定的缺血时间后 ,使血管再通 ,建立心肌的缺血再灌注模型。采用透射电镜、原位末端探针标记 ,图像分析 ,对缺血再灌注损伤区心肌细胞的损伤情况进行研究。结果　电镜观察显示缺血再灌注损伤区呈现明显的心肌细胞凋亡征象 ,TUNEL检测结果显示缺血再灌注损伤区出现心肌凋亡阳性细胞 ,平均凋亡率为 2 1 10 %。结论　心肌缺血再灌注损伤的发生与凋亡机制有关 ,并可能是其发生的重要原因。     

不同时段的心肌缺血后再灌注损伤与心肌细胞凋亡的实验研究

目的　探讨实验性不同时段的心肌缺血后再灌注损伤是否致心肌细胞凋亡、凋亡率的大小及不同的缺血时间与凋亡率的关系。方法　阻断左冠状动脉的左室支建立心肌缺血模型 ,达到预定的缺血时间后 ,使血管再通 ,建立心肌的缺血再灌注模型。采用透射电镜、原位末端探针标记、图像分析和统计处理对缺血灌注损伤区心肌细胞的损伤情况进行研究。结果　电镜观察和原位未端标记 (TUNEL)检测发现在经历了不同的缺血时间后 ,再灌注均可致心肌细胞凋亡 ,但不同的缺血时间后再灌注致心肌细胞的凋亡率不同 ,实验Ⅰ～Ⅲ组平均凋亡率分别为 3 2 7%、18 4 3%和 2 0 2 8%。缺血时间越长凋亡率越高。结论　心肌缺血再灌注损伤的发生与凋亡机制有关 ,且凋亡率的大小在一定的时间段内可能与心肌的缺血时间呈正相关     

血塞通预处理对缺血再灌注损伤大鼠心肌细胞凋亡的影响

目的探讨血塞通注射液对缺血再灌注损伤(MIRI)大鼠心肌细胞凋亡的影响。方法采用脱氧核糖核苷酸转移酶介导的脱氧尿嘧啶缺口末端标记法原位检测心肌细胞凋亡。透射电镜观察缺血损伤区心肌,摄片检测心肌细胞凋亡。结果X组和MIR组心肌细胞凋亡指数(AI)较S组显著增高(P<0.001,P<0.05),X组AI较MIR组低(P<0.05)。电镜下对照组未发现细胞凋亡,而在MIR组细胞凋亡明显增多,X组心肌细胞凋亡特征较MIR组明显减少。结论血塞通能减轻MIRI对大鼠心肌毒性作用,抑制心肌细胞凋亡,达到心肌保护作用。     

卡托普利预处理对家兔实验性心肌梗塞范围及心肌细胞凋亡的影响

目的:探讨卡托普利预处理对家兔实验性心肌梗塞范围及心肌细胞凋亡的影响。方法:健康家兔,分为(1)假手术对照组(sham-operated control group),(2)急性心梗组(acuteinfarct group),(3)卡托普利预处理组(captopril group,25mg·kg^-1·d^-1,喂饲1周)。结扎冠状动脉左旋支造成急性心肌梗塞模型。测结扎前、结扎后15、30、60min的心功能。结扎前及结扎后1h的全血粘度与血球压积。结扎后6h取心脏,以缺血区重/左室重比为缺血范围,梗塞区重/缺血区重比为梗塞范围。TUNEL法染色检测细胞凋亡,以心肌凋亡阳性细胞数占总心肌细胞数的百分比作为心肌细胞凋亡指数。结果:(1)卡托普利预处理组梗塞范围显著小于心梗组(16.60%±0.94%比36.24%±1.94%,P＜0.05),并改变左室功能及血粘度。(2)梗塞周围可见凋亡心肌细胞,卡托普利预处理该区心肌细胞凋亡指数明显小于急性心梗组(26.37%±0.71%比42.44%±2.32%,P＜0.05)。结论:卡托普利预处理可明显减少梗塞范围及心肌细胞凋亡程度,改善心功能。     

急性缺血诱导大鼠心肌细胞凋亡的时间演变和空间分布

目的:探讨急性缺血诱导大鼠心肌细胞凋亡的时间演变和空间分布规律。方法:以DNA电泳和TUNEL分析检测SD大鼠心肌的细胞凋亡情况。结果:缺血2 h开始出现DNA梯形条带和TUNEL阳性细胞,DNA条带和凋亡指数随缺血时间延长而显著增加(P<0.01)。TUNEL阳性心肌细胞在缺血区散在分布,缺血边缘区凋亡指数显著高于缺血中央区(P<0.05),缺血区内膜下凋亡指数显著高于外膜下(P<0.01)。结论:在本实验设定的时间范围内,急性缺血诱导的心肌细胞凋亡与缺血时间具有时效关系;调亡的心肌细胞在缺血区散在分布,以缺血区两侧边缘和内膜下心肌多见。     

缺血预适应对青年与老年大鼠缺血/再灌注心肌氧化应激及线粒体功能的影响

目的:探讨缺血预适应(ischemic preconditioning,IPC)对青年与老年大鼠缺血/再灌注(ischemia/reperfusion,IR)心肌损伤的影响。方法:雄性3月龄(青年)与20月龄(老年)Wistar大鼠,应用离体心脏灌流方法复制心肌IR与IPC模型。实验分为青年缺血/再灌注(YIR)组、青年缺血预适应(YPC)组、老年缺血再灌注(OIR)组与老年缺血预适应(OPC)组。透射电镜观察心肌及心肌线粒体超微结构变化;TTC染色测定心肌梗死面积;比色法测定冠脉流出液中乳酸脱氢酶(LDH)活性、心肌组织中超氧化物歧化酶(SOD)活性及丙二醛(MDA)含量;酶联免疫吸附法测定心肌组织硝基化与羰基化蛋白质含量,TUNEL方法检测心肌细胞凋亡;氧电极法测定线粒体呼吸功能及钙诱导的线粒体渗透性转运孔开放情况。结果:与YIR组比较,YPC组心肌梗死面积明显减少,冠脉流出液中LDH活性降低,心肌组织的SOD活性增加,MDA含量降低,心肌硝基化与羰基化蛋白含量降低。电镜下可见YPC组心肌及分离的心肌线粒体膜结构完整、基质致密。YIR组心肌线粒体呼吸控制率与Ⅲ态耗氧量及P/O比值均明显增加,质子漏出减少,钙诱导的线粒体肿胀明显减轻,心肌细胞凋亡率下降。而与OIR组比较,OPC组上述指标均无显著统计学差异。与YPC组比较,OPC组心肌超微结构损伤明显增加,心肌氧化应激水平增加,线粒体呼吸功能下降,心肌细胞凋亡与坏死增多。结论:缺血预适应能够保护青年大鼠心肌缺血/再灌注损伤;而老年大鼠心脏对预适应刺激减敏,导致缺血预适应心肌保护作用钝化,这可能与老龄IPC心脏氧化应激水平增加导致线粒体损伤、细胞凋亡有关。     

大鼠实验性心肌缺血再灌注时心肌细胞凋亡的动态变化及意义

探讨大鼠心肌缺血再灌注时心肌细胞凋亡的动态变化及其与心肌缺血再灌注损伤的相互关系。结果显示假手术组与心肌缺血１５ｈ未见心肌细胞凋亡,但缺血３５ｈ或缺血３０ｍｉｎ再灌注ｌｈ已见有细胞凋亡,细胞凋亡出现明显早于细胞坏死。细胞凋亡指数（ＡＩ）随缺血或再灌注时间延长呈渐增趋势,分别以缺血４８．５ｈ（４９８％士１７．２７％）和缺血３０ｍｉｎ再灌注４８ｈ（３８．１５％±１３．２６％）最高。与心肌缺血组比较,心肌缺血再灌注组相同处理时间的大鼠的ＡＩ较低（Ｐ＜０．０５）。凋亡细胞主要位于心肌纤维收缩带区域及心肌梗死区周围。电镜示再灌注;０肌组织中有典型细胞凋亡形态改变。研究提示心肌细胞凋亡是持续性心肌缺血中早期心肌细胞死亡的主要形式,再灌注过程虽减少凋亡细胞数量,但却促进了不可逆损伤的心肌细胞的凋亡过程。     

脑皮质微血管梗塞模型中的迟发性神经元死亡

目的 :探讨脑皮质微血管梗塞后迟发性神经元死亡的发生机制。方法 :应用光化学反应原理 ,静脉注射光敏染科孟加拉红 ,绿色激光透过颅骨对大鼠脑皮质感觉运动区作定向照射 ,形成脑皮质微血管梗塞模型 ,进行神经功能评分、TTC和HE染色光镜观察、透射电镜观察和原位末端标记。结果 :脑皮质微血管梗塞后 48h ,神经功能缺损发展至高峰 ,神经元和内皮细胞损伤最为严重 ,梗塞灶中心神经元以坏死为主 ,半暗带区凋亡细胞数目显著增加。结论 :光化学法诱导大鼠脑皮质微血管梗塞模型中皮质神经元迟发性死亡呈一定的时间和空间分布特征 ,缺血半暗带神经元凋亡与这种延迟损伤有关     

New directions in strategies using cell therapy for heart disease

Congestive heart failure remains a major public health problem and is frequently the end result of cardiomyocyte apoptosis and fibrous replacement after myocardial infarction, a process referred to as left ventricular remodeling. Cardiomyocytes undergo terminal differentiation soon after birth and are generally considered to irreversibly withdraw from the cell cycle. In response to ischemic insult adult cardiomyocytes undergo cellular hypertrophy, nuclear ploidy, and a high degree of apoptosis. A small number of human cardiomyocytes retain the capacity to proliferate and regenerate in response to ischemic injury. However, whether these cells are derived from a resident pool of cardiomyocyte stem cells or from a renewable source of circulating bone marrow-derived stem cells that home to the damaged myocardium is at present not known. Replacement and regeneration of functional cardiac muscle after an ischemic insult to the heart could be achieved by either stimulating proliferation of endogenous mature cardiomyocytes or resident cardiac stem cells or by implanting exogenous donor-derived or allogeneic cells such as fetal or embryonic cardiomyocyte precursors, bone marrow derived mesenchymal stem cells, or skeletal myoblasts. The newly formed cardiomyocytes must integrate precisely into the existing myocardial wall in order to augment synchronized contractility and avoid potentially life-threatening alterations in the electrical conduction of the heart. A major impediment to survival of the implanted cells is altered immunogenicity by prolonged ex vivo culture conditions. In addition, concurrent myocardial revascularization is required to ensure viability of the repaired region and prevent further scar tissue formation. Human adult bone marrow contains endothelial precursors which resemble embryonic angioblasts and can be used to induce infarct bed neovascularization after experimental myocardial infarction. This results in protection of cardiomyocytes against apoptosis, induction of cardiomyocyte proliferation and regeneration, long-term salvage and survival of viable myocardium, prevention of left ventricular remodeling, and sustained improvement in cardiac function. It is reasonable to anticipate that cell therapy strategies for ischemic heart disease will need to incorporate (a) a renewable source of proliferating, functional cardiomyocytes, and (b) angioblasts to generate a network of capillaries and larger size blood vessels for supply of oxygen and nutrients to both the chronically ischemic endogenous myocardium and to the newly implanted cardiomyocytes     

Interleukin-6/soluble interleukin-6 receptor complex reduces infarct size via inhibiting myocardial apoptosis

Apoptosis of cardiomyocytes plays an important role in reperfusion injury following myocardial infarction. Conversely, interleukin-6 (IL-6)--a potent cytokine--inhibits myeloma cell apoptosis by activating GP130 through the IL-6 receptor (IL-6R). We hypothesized that the IL-6/soluble IL-6R complex can inhibit myocardial apoptosis, and limit infarct size in reperfused acute myocardial infarction. Anesthetized rats were randomly divided into five groups: sham, coronary occlusion and reperfusion rats administered IL-6/soluble IL-6R complex, IL-6 alone, soluble IL-6R (sIL-6R) alone, or a control vehicle. Rats were subjected to 30 min occlusion of the left coronary artery followed by 3 h reperfusion. After reperfusion, the hearts were excised. For detection and quantification of apoptosis, gel electrophoresis of extracted genomic DNA and TUNEL method of paraffin sections were performed. The percentage of the infarct area was measured using tetrazolium chloride staining. The cardiomyocyte apoptosis analysis revealed that apoptosis in the reperfused myocardium was inhibited only in the complex group. Furthermore, the percentage of the infarct area out of the area at risk was remarkably reduced in the complex group (23.8+/-1.8%), compared with that in the vehicle (37.9+/-3.7%), the IL-6 (40.7+/-1.0%), or the sIL-6R (37.5+/-2.4%) groups (P=0.0002). No significant differences were observed among the vehicle, IL-6, and sIL-6R groups. The IL-6/soluble IL-6 receptor complex inhibits cardiomyocyte apoptosis in reperfused acute myocardial infarction. It possibly reduces irreversible reperfusion injury.     

急性心肌梗死晚期再灌注后心肌细胞凋亡与Bcl-2、Bax蛋白表达的研究

目的： 探讨犬急性心肌梗死后晚期再灌注对梗死周边缺血区心肌细胞凋亡及凋亡相关蛋白Bcl-2、Bax表达的影响。方法: 健康成年杂交犬28只，全麻下常规开胸暴露冠状动脉后随机分为3组：假手术组（n=8）,急性心肌梗死组（n=10）、晚期再灌注组（n=10）。假手术组仅行左冠状动脉前降支下穿过丝线而不结扎冠状动脉，急性心肌梗死组行左冠状动脉前降支高位永久结扎，晚期再灌注组在高位结扎左冠状动脉前降支 6 h 后松解结扎线予以再灌注 6 h。共有23只犬模型制作成功。各组犬均于术后 12 h 处死，采集心肌标本。使用TUNEL法检测心肌细胞凋亡，免疫组化染色和Western blotting蛋白印迹分析Bcl-2、Bax在心肌细胞中表达情况。结果: 晚期再灌注组心肌细胞凋亡数较急性心肌梗死组明显减少（P<0.05），但两组心肌细胞凋亡数均高于假手术组（P<0.01）。与假手术组相比，急性心肌梗死组和晚期再灌注组Bcl-2蛋白的表达均升高（P<0.01），其中在晚期再灌注组的表达略多于急性心肌梗死组，但无显著差异（P＞0.05）。Bax蛋白在晚期再灌注组的表达高于假手术组（P<0.01），但低于急性心肌梗死组（P<0.05）。结论: 急性心肌梗死后晚期再灌注可以减少梗死周边缺血区心肌细胞凋亡，其机制可能与心肌细胞表达Bax蛋白减少有关。     

急性心肌梗死晚期再灌注后心肌细胞凋亡与Bcl-2、Bax 蛋白表达的研究

目的:探讨犬急性心肌梗死后晚期再灌注对梗死周边缺血区心肌细胞凋亡及凋亡相关蛋白Bcl-2、Bax表达的影响。方法:健康成年杂交犬28只,全麻下常规开胸暴露冠状动脉后随机分为3组:假手术组(n=8),急性心肌梗死组(n=10)、晚期再灌注组(n=10)。假手术组仅行左冠状动脉前降支下穿过丝线而不结扎冠状动脉,急性心肌梗死组行左冠状动脉前降支高位永久结扎,晚期再灌注组在高位结扎左冠状动脉前降支6 h后松解结扎线予以再灌注6 h。共有23只犬模型制作成功。各组犬均于术后12 h处死,采集心肌标本。使用TUNEL法检测心肌细胞凋亡,免疫组化染色和Western blotting蛋白印迹分析Bcl-2、Bax在心肌细胞中表达情况。结果:晚期再灌注组心肌细胞凋亡数较急性心肌梗死组明显减少(P<0.05),但两组心肌细胞凋亡数均高于假手术组(P<0.01)。与假手术组相比,急性心肌梗死组和晚期再灌注组Bcl-2蛋白的表达均升高(P<0.01),其中在晚期再灌注组的表达略多于急性心肌梗死组,但无显著差异(P>0.05)。Bax蛋白在晚期再灌注组的表达高于假手术组(P<0.01),但低于急性心肌梗死组(P<0.05)。结论:急性心肌梗死后晚期再灌注可以减少梗死周边缺血区心肌细胞凋亡,其机制可能与心肌细胞表达Bax蛋白减少有关。     

Relationship between Apoptosis and Expression of Heat Shock Proteins in Peripheral Blood Lymphocytes of Patients with Myocardial Infarction

We studied activity and dynamics of apoptosis of peripheral blood lymphocytes in patients with myocardial infarction and analyzed the relationship of these processes with expression of heat shock proteins with a molecular weight of 70 kDa playing an essential role in preventing cell death. Thus, we first demonstrated activation of apoptosis in peripheral blood cells of patients with myocardial infarction compared to the control (healthy individuals) and revealed the expected negative correlation between the expression of heat shock proteins with a molecular weight of 70 kDa by lymphocytes and intensity of their death. The observed dynamics of mononuclear cell apoptosis in the peripheral blood of patients with myocardial infarction can reflect activity of programmed cardiomyocyte death in the focus of ischemic injury.     

Role of CD11b+Gr-1+ myeloid cells in AGEs-induced myocardial injury in a mice model of acute myocardial infarction

Aims: Polymorph neutrophils are the predominant inflammatory cells and play a crucial role on the pathogenesis of myocardial injury at the early stage of acute myocardial infarction (AMI). However, the precursors and the differentiation of neutrophils are not fully understood. Here we explored the role of CD11b⁺Gr-1⁺ myeloid-derived suppressor cells (MDSCs) on myocardial injury in the absence and presence of advanced glycation end-products (AGEs) in a mice model of AMI. Methods and Results: Male C57BL/6J mice were selected. Fluorescent actived cell sortor (FACS) data demonstrated significantly increased CD11b⁺Gr-1⁺ MDSCs both in peripheral blood circulation and in the ischemic myocardium at 24 hours post AMI. Quantitative-real-time PCR results also revealed significantly upregulated CD11b and Ly6G mRNA expression in the ischemic myocardium. AGEs treatment further aggravated these changes in AMI mice but not in sham mice. Moreover, AGEs treatment also significantly increased infarction size and enhanced cardiomyocyte apoptosis. The mRNA expression of pro-inflammatory cytokine IL-6 and iNOS2 was also significantly increased in AMI + AGEs group compared to AMI group. Conclusion: These data suggest enhanced infiltration of MDSCs by AGEs contributes to aggravated myocardial injury in AMI mice, which might be one of the mechanisms responsible for severer myocardial injury in AMI patients complicating diabetes.