β_3肾上腺素能受体在大鼠缺血心肌中的表达

目的探究β_3肾上腺素能受体在大鼠缺血心肌中的表达。方法结扎大鼠冠状动脉建立心肌慢性缺血模型,应用免疫组化和Western blot检测β_3受体在缺血后心肌中的表达及分布。结果免疫组化结果显示,与假手术组对比,手术组中慢性缺血心肌细胞有强β_3受体蛋白表达信号,其分布不均一。Western blot提示,手术组大鼠心肌β_3受体表达量明显高于假手术组,差异有统计学意义(P0.001)。心肌组织中的微血管内皮细胞也有β_3受体蛋白表达。结论大鼠慢性缺血心肌组织中的心肌细胞和心肌微血管内皮细胞β3受体表达增多。     

脂肪酸结合蛋白评价大鼠缺血性心肌损伤的价值

目的探讨脂肪酸结合蛋白(H-FABP)在异丙肾上腺素诱导的心肌缺血中对评价心肌损伤中的价值。方法将128只大鼠随机分成4组,采用异丙肾上腺素建立不同程度的心肌缺血模型,分为正常对照组和低、中、高剂量实验组,取4个时间点,用Western印迹法检测不同程度心肌缺血大鼠血清中H-FABP表达水平,分析心肌缺血后血清H-FABP含量与时间关系,分析大鼠心肌缺血严重程度与血清H-FABP含量之间关系。结果 1心肌缺血大鼠模型建立成功,大鼠心肌缺血后血清中H-FABP含量2 h已有升高,4 h明显增高,与正常对照组差异显著(P0.05),16 h已有下降,32 h基本降至正常。2大鼠血清H-FABP升高的倍数随其心肌缺血程度加重而递增(P0.05)。结论 H-FABP在心肌缺血发生的早期即有明显升高,持续时间较短,H-FABP可作为缺血心肌损伤早期诊断的心肌标志物和评价损伤程度指标。     

急性缺血心肌脂肪酸代谢的变化

目的 观察急性缺血心肌心脏型脂肪酸结合蛋白(H-FABP)表达及血清游离脂肪酸(FFA)含量的动态变化.方法 健康SD雄性大鼠随机分为对照组和5个实验组(实验1、2、 3、4、5组分别为心肌缺血1、2、4、6、12 h);HE染色观察各实验组心肌缺血后病理形态学改变,用Western印迹检测心肌H-FABP表达的变化,用一步提取比色法测定血清FFA含量.结果 (1)缺血2、4 h组心肌细胞明显肿胀,有部分形成了"收缩带";缺血6、12 h心肌细胞边界不清、局灶性凝集坏死,缺血12 h组有大量炎性细胞浸润.(2)Western印迹法检测: 实验2、3、4、5组与对照组比较心肌H-FABP蛋白均表达下降,实验4组降低约41%(P＜0.01).(3)FFA测定:实验2、3、4、5组与对照组比较大鼠血清FFA含量均不同程度升高,尤以缺血2、4 h组为甚(P＜0.01),分别升高约2.01倍和2.72倍.结论 急性心肌缺血后H-FABP表达明显下降,血清FFA含量明显升高,可能导致心肌细胞代谢紊乱,促进心肌肥厚重构.     

缺血心肌细胞骨架损伤研究

目的建立动物心肌缺血模型，探讨心肌缺血时细胞骨架的改变。方法15只清洁级健康成年雄性Wistar大白鼠，采用左冠状动脉结扎术造成部分心肌缺血，分别于缺血30min、60min、120min时取缺血心肌组织用免疫组织化学方法观察心肌细胞骨架蛋白肌动蛋白（α-actin）、波形蛋白（vimentin）、肌球蛋白（myosin）、结蛋白（desmin）的变化，与未缺血部分心肌对比，并用计算机图像模拟分析系统计算细胞骨架蛋白量的变化。结果心肌缺血时30min时即有骨架蛋白actin、myosin的损伤，随后见desmin、vimentin受损。结论心肌缺血时可以早期导致心肌细胞的细胞骨架损伤。     

结蛋白与心脏疾病

心肌细胞结构和功能的完整有赖于由微丝，微管和中间丝组成的分布广泛的细胞骨架蛋白，结蛋白是心肌中主要的中间丝蛋白，对于维持心肌细胞结构和功能有重要的作用。结蛋白缺失破坏会导致心肌收缩力降低，导致各种心脏疾病的发生。本文就心脏疾病中结蛋白的变化及其对心功能的影响做一综述。     

不同缺血时段大鼠心肌组织中Nanog的表达变化及意义

目的观察不同缺血时段大鼠心肌组织中Nanog的表达变化,并探讨其意义。方法健康雄性SD大鼠30只,随机分为假手术组（正常组）和Q1、Q2、Q3、Q4组,每组6只,后四组分别在心肌缺血2、6、12、24 h取心肌组织,采用RT-PCR和Western blot方法观察各组大鼠心肌组织中Nanog mRNA和蛋白表达变化。结果正常组和Q1、Q4组心肌组织中Nanog mRNA和蛋白表达最低,Q2、Q3组表达最高（P均〈0.05）。结论缺血6～12 h大鼠心肌组织中Nanog mRNA和蛋白表达最高;Nanog可能在缺血后的心肌重构过程中发挥作用。     

左旋卡尼汀对兔心肌缺血再灌注损伤的保护

目的探讨左旋卡尼汀在心肌缺血再灌注损伤状态下对心肌的保护作用。方法制备兔缺血再灌注模型,实验分为空白对照组、盐水对照组和左旋卡尼汀组,左旋卡尼汀组心肌缺血30 min后给予左旋卡尼汀。观察各组缺血再灌注过程中心电图的动态改变,以及再灌注结束后动脉血游离脂肪酸、超氧化物歧化酶、丙二醛、肌酸激酶的含量和组织中Na -K -ATP酶和Ca2 -Mg2 -ATP酶活性;用Western blot法检测结扎点以下5 mm处左心室全层心肌热休克蛋白70的含量。结果盐水对照组和左旋卡尼汀组均造成明显的心电图动态改变,与盐水对照组比较,左旋卡尼汀组心电图ST段出现有效改善;左旋卡尼汀组分别与盐水对照组和空白对照组相比,游离脂肪酸和丙二醛含量均显著减少(P<0.05);Na -K -ATP酶、Ca2 -Mg2 -ATP酶活性及超氧化物歧化酶的含量显著增多(P<0.05),肌酸激酶含量有下降趋势(P>0.05);心肌热休克蛋白70含量显著增多(P<0.05)。结论左旋卡尼汀可能诱导产生热休克蛋白70,并对心肌缺血再灌注损伤有保护作用。     

老年大鼠沉默信息调节因子相关酶1的变化及其与心肌缺血预处理“失效”的关系

目的 通过观察成年和老年大鼠内源性及心肌缺血预处理沉默信息调节因子1(SIRT1)的差异,在与寿命相关的SIRT1蛋白水平揭示缺血预处理在老年大鼠中效果较成年大鼠差的可能原因.方法 雄性SD大鼠60只,成年和老年各30只,其中成年大鼠200～ 250 g,老年大鼠450～ 600 g(18月龄).两个年龄组分别采用随机数字表法随机分为3组(n=10),假手术(S组),缺血再灌注组(IR组),缺血预处理组(IPC组).通过结扎及松开冠状动脉左前降支建立在体大鼠缺血预处理及缺血再灌注模型,分别于缺血前及再灌注120 min监测血流动力学;分光光度法测定心肌组织中乳酸脱氢酶(LDH)含量、肌酸激酶同工酶(CK-MB)含量;ELISA法检测心肌组织SIRT1活性;RT-PCR法检测心肌组织中SIRT1蛋白mRNA;Western蛋白印迹检测SIRT1蛋白含量.结果 老年大鼠SIRT1蛋白基础水平较成年大鼠降低,SIRT1基础活性亦较成年大鼠下降(P＜0.05).缺血预处理时老年大鼠SIRT1含量较成年大鼠显著降低,SIRT1活性显著降低(P＜0.05).老年大鼠缺血预处理与老年大鼠缺血再灌注相比,SIRT1含量与活性均无显著改变(P＜0.05).成年大鼠缺血预处理与成年大鼠缺血再灌注相比,SIRT1含量与活性均显著升高.结论 SIRT1含量及活性随年龄增加而降低是缺血预处理在老年大鼠中丧失保护作用的原因.     

2型糖尿病患者血清对人脐静脉内皮细胞骨架及胞浆内游离钙的影响

目的 探讨2型糖尿病患者血清干预人脐静脉内皮细胞(ECV-304)对细胞骨架微丝及胞浆内游离钙浓度的影响.方法 ECV-304传代后取对数生长期细胞分为正常对照组(N组)、健康人血清干预组(H组)和糖尿病患者血清干预组(MD组).按照1×105/ml将细胞种植在Petri 皿中进行分组干预,其中健康人血清、糖尿病患者血清浓度分别占总体积的10%.采用Fluo-3/AM作为荧光指示剂在激光扫描共聚焦显微镜下观察细胞内游离钙浓度的变化,采用荧光探针标记的鬼笔环肽染色法观察细胞骨架微丝分布的差异.结果 与N组比较MD组胞浆内荧光分布不均匀,胞浆内游离钙浓度显著升高,骨架微丝断裂,排列紊乱,少部分区域缺失.结论 糖尿病患者血清干预正常的ECV-304可以造成内皮细胞的骨架结构改变,主要与细胞内游离钙浓度升高有关.     

Bnip3及其介导的线粒体自噬在大鼠心肌缺血再灌注过程中作用的研究

目的从在体动物水平和整体生理学角度,探索Bnip3及线粒体自噬与心肌缺血再灌注发生时心肌组织损伤程度、心肌梗死面积大小以及心室功能障碍的关系。方法雄性SD大鼠35只,通过结扎大鼠心脏冠状动脉左前降支构建心梗模型,分为假手术组、缺血30min组、缺血再灌注组分为再灌注15min、30min、60min组。术中监测血流动力学指标以评价左室功能变化,Wester n blot定量分析Bnip3蛋白在大鼠心肌缺血再灌注不同时期的表达水平,HE染色、免疫组化(LC-3、BCL-1)及电镜观察心肌组织损伤程度和心肌细胞中线粒体自噬水平。结果心肌缺血再灌注早期(15-60min)大鼠血压明显下降(P0.05),心肌组织出现不可逆损伤,心肌细胞中出现线粒体自噬。Bnip3蛋白表达量在再灌注早期(30、60min)显著下降(P0.5)。结论 Bnip3介导的线粒体自噬在心肌缺血再灌注早期的病理生理改变中起到重要作用。     

Disruption of microtubules as an early sign of irreversible ischemic injury. Immunohistochemical study of in situ canine hearts.

Structural disruption of the cytoskeleton may be involved in irreversible ischemic injury. In the present study, ischemic changes in microtubules during various periods of myocardial ischemia were studied with an immunohistochemical technique in open-chest dogs. In intact myocardium, microtubules were stained as a filamentous network throughout cytoplasm and a circular network around the nucleus, which disappeared with colchicine treatment. In brief ischemia of less than 15 minutes, microtubule patterns were unaltered. After 20 minutes, however, characteristic microtubule stains were partly lost in patchy lesions. As an increase in ischemic period, lesions of loss of microtubule stains were increased in number and size. After 120 minutes of reperfusion following 60 minutes of ischemia, the lesions with intact actin filaments but with disrupted microtubules were replaced by the severely injured cells in which the regular myofibrillar registrations were distinctly disrupted. After 24 hours of reperfusion following 40 minutes of occlusion of the left circumflex artery, the percent area of disrupted microtubules at 40 minutes of ischemia was replaced by that of irreversibly injured lesions in the posterior papillary muscle. These results indicate that disruption of microtubules during ischemia heralds irreversible ischemic injury. However, in in vitro study, the myocardium incubated in hypoxic solution for 60-120 minutes demonstrated earlier disruption of the microtubules than the vinculin. Electron microscopic study also showed minimal irreversible changes in the lesions with disrupted microtubules. Thus, taken together, we conclude that microtubules that support the structural integration of myofibrils and other organelles are disrupted in severe myocardial ischemia before the irreversible injury, promoting the irreversible change after reperfusion.(ABSTRACT TRUNCATED AT 250 WORDS)     

beta-Dystroglycan modulates the interplay between actin and microtubules in human-adhered platelets

To maintain the continuity of an injured blood vessel, platelets change shape, secrete granule contents, adhere, aggregate, and retract in a haemostatic plug. Ordered arrays of microtubules, microfilaments, and associated proteins are responsible for these platelet responses. In full-spread platelets, microfilament bundles in association with other cytoskeleton proteins are anchored in focal contacts. Recent studies in migrating cells suggest that co-ordination and direct physical interaction of microtubules and actin network modulate adhesion development. In platelets, we have proposed a feasible association between these two cytoskeletal systems, as well as the participation of the dystrophin-associated protein complex, as part of the focal adhesion complex. The present study analysed the participation of microtubules and actin during the platelet adhesion process. Confocal microscopy, fluorescence resonance transfer energy and immunoprecipitation assays were used to provide evidence of a cross-talk between these two cytoskeletal systems. Interestingly, beta-dystroglycan was found to act as an interplay protein between actin and microtubules and an additional communication between these two cytoskeleton networks was maintained through proteins of focal adhesion complex. Altogether our data are indicative of a dynamic co-participation of actin filaments and microtubules in modulating focal contacts to achieve platelet function.     

曲美他嗪对心肌缺血时细胞骨架损伤的作用

目的　探讨心肌缺血时细胞骨架改变并观察曲美他嗪对心肌缺血时细胞骨架损伤的影响 ,为临床应用提供理论依据。方法　将大白鼠随机分为对照组和用药组并制成缺血模型 ,分别于缺血 30 m in、6 0 min、12 0 min时取心肌组织用免疫组化的方法观察肌动蛋白、波形蛋白、肌球蛋白、结蛋白等心肌细胞骨架蛋白的改变 ,并用计算机图象模拟分析系统计算骨架蛋白量的变化。结果　心肌缺血 30 min即有肌动蛋白、肌球蛋白损伤 ,12 0 min时有结蛋白损伤 (P<0 .0 5 )。曲美他嗪干预后 ,心肌缺血 6 0 min、12 0 min时肌动蛋白、肌球蛋白损伤明显减少 (P<0 .0 5 )。结论　心肌缺血时可以导致细胞骨架损伤 ,曲美他嗪对心肌缺血时细胞骨架损伤有保护作用     

细胞骨架在原代培养乳鼠窦房结细胞模拟缺血预适应中的作用研究

观察细胞骨架在乳鼠窦房结细胞模拟缺血预适应(IP)中的作用。取培养 2d的乳鼠窦房结细胞,随机分为①对照组;②模拟缺血 /再灌注(I/R)组;③模拟IP组;④phalloidin(微丝聚合剂 ) +I/R组;⑤cytochalasinD(微丝解聚剂) +IP组:⑥Taxol(微管聚合剂 ) +I/R组;⑦colchicine(微管解聚剂 ) +IP组。以FITC phalloidin及SABC cy3试剂盒分别标记F actin及β tubulin,用激光共聚焦显微镜检测各组窦房结细胞荧光强度改变。结果:①phalloi din预处理能显著增强再灌注后窦房结细胞F actin及β tubulin的荧光强度,并维持其形态、结构的相对完整性,模拟IP效应。②cytochalasinD及colchicine均能阻断模拟IP的细胞骨架保护作用,显著降低窦房结细胞F actin及β tubulin的荧光强度,导致细胞骨架的解体。③Taxol未能对I/R窦房结细胞提供IP样保护作用。结论:维持微丝结构的相对完整性可以减轻窦房结细胞I/R损伤,模拟IP效应;维持细胞骨架的相对完整性是IP产生的重要前提。     

Flow cytometric analysis of isolated adult cardiomyocytes: vinculin and tubulin fluorescence during metabolic inhibition and ischemia.

Immunofluorescence and quantitative flow cytometry was used to determine if alterations in cytoskeletal proteins (vinculin and tubulin) occur during metabolic inhibition and ischemic incubation of isolated adult rat cardiomyocytes. Effects of cell shape changes on fluorescence, were controlled for by the contractile inhibitor, butanedione monoxime (BDM) and gated analysis. Flow cytometry differentiated rod- and round-shaped myocytes on the basis of forward and side scattering. Severe contracture of metabolically inhibited (iodoacetic acid and amytal) myocytes caused an artefactual increase in fluorescence intensity and a redistribution of tubulin into microblebs on the cell surface, which tended to mask specific losses of fluorescence. Fluorescence microscopy showed that round cells stained intensely for vinculin, but not for tubulin and that vinculin redistributed into coarse patches between 60 and 90 min, times which corresponded to small rebounds of fluorescence. With gated analysis, to exclude severely contracted round and squared cells, and with BDM inhibition of contracture, both metabolically inhibited and ischemic pelleted myocytes showed an early decrease in specific immunofluorescence staining for tubulin and vinculin, which preceded loss of cell viability, as determined by trypan blue staining. In both ischemic and metabolically inhibited cells, decreases of vinculin fluorescence preceded or coincided with increasing osmotic fragility. It is concluded that early cytoskeletal alterations of vinculin in ischemic and anoxic injury correlate with the development of osmotic fragility and irreversible myocyte injury.     

预处理对在体大鼠心肌细胞的保护作用

目的：探讨缺血预处理对心肌细胞的保护作用。方法：在体大鼠心肌细胞缺血／复灌模型中，观察缺血预处理对心肌细胞再次长时间缺氧／复氧或缺血／复灌损伤的保护作用（LDH释放，MDA，SOD的含量和心肌梗死范围及心律失常等）。结果；在体大鼠心肌缺血预处理组梗死范围和血清LDH较缺血复灌组减少（P＜0．01），预处理组MDA较缺血复灌组也显著降低（P＜0．01）。而且无论是再次的缺血还是复灌期室性心律失常的发生率预处理组明显低于非预处理组（P＜0．01）。结论：缺血预处理可以减少再次长时间的缺血／复灌对心肌的损伤。     

肌动蛋白、肌钙蛋白及纤维连接蛋白在心肌缺血中的研究

目的探讨肌动蛋白（actin，克隆号HHF35）、肌钙蛋白T（cTnT）和纤维连接蛋白（fibronectin，Fn）在缺血心肌的表达情况。方法采用第二代EliVision TM免疫组化方法、图像分析技术检测HHF35、cTnT及Fn在正常心脏、急性心肌梗死和可疑缺血心肌的人尸检标本中的表达情况。结果（1）HHF35、cTnT、Fn在急性心肌梗死组、可疑心肌缺血组与正常心脏组的表达间差别均有显著性意义（P〈0．01），而急性心肌梗死组与可疑心肌缺血组之间差异亦有显著性意义（P〈0．05）；（2）在急性心肌梗死组、可疑心肌梗死组，Fn与HHF35、cTnT呈正相关，相关系数分别为0．496、0．527（P〈0．05）。结论肌动蛋白、肌钙蛋白T和纤维连接蛋白可能成为人心肌缺血的诊断指标。     

Cytoskeletal damage during myocardial ischemia: changes in vinculin immunofluorescence staining during total in vitro ischemia in canine heart

The role of cytoskeletal damage in the disruption of the plasma membrane observed during myocardial ischemia has been studied using antibodies to vinculin to identify changes in the distribution of this membrane associated cytoskeletal protein. Vinculin is a component of the cytoskeletal attachment complex between the plasma membrane and the Z-line of the underlying myofibrils. The effects of varying periods of total ischemia on the localization of vinculin were assessed by immunofluorescence and evidence of membrane disruption was evaluated by electron microscopy. Thin tissue slices prepared from the ischemic tissue were incubated in oxygenated Krebs-Ringer phosphate buffer at 37 degrees C to assess inulin permeability, ultrastructure, and any changes in the distribution of vinculin associated with incubation. The previously reported costameric pattern of vinculin staining was observed in longitudinal sections of control myocardium, myocardium subjected to 60 minutes of total ischemia, and myocardium subjected to 60 minutes of ischemia followed by 60 minutes of incubation in oxygenated media. Electron microscopy and inulin permeability measurements confirmed that plasma membrane integrity was preserved under these conditions. However, when the duration of total ischemia was extended to 120 minutes or longer, there was a progressive loss of vinculin staining along the lateral margin of myocytes. This change correlates with the appearance of subsarcolemmal blebs and breaks in the plasma membranes observed by electron microscopy and confirmed by the increase in inulin permeability observed in tissue slices.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modification of actin,myosin and tubulin distribution during cytoplasmic granule movements associated with platelet adhesion

BACKGROUND AND OBJECTIVES: Cytoskeletal elements determine the changes in platelet cell shape which occur during adhesion, aggregation and release of granular contents as part of the activation process. The aim of this study was to characterize the changes in the distribution of actin filaments, myosin and tubulin molecules during several stages of platelet adhesion to glass and their association with granule displacement, as assessed by confocal microscopy. DESIGN AND METHODS: Platelets obtained from healthy donors were adhered to glass and cytoskeleton distribution was characterized and correlated to changes of cell shape and intracellular granule displacement by immunofluorescence assays and phase contrast microscopy. Treatment with specific cytoskeleton inhibitors such as cytochalasin D, butanedione monoxime and colchicine were used before and after the adhesion process. The spatial distribution of the cytoskeleton in association with cytoplasmic granules was analyzed in both confocal microscopy projections and three-dimensional images obtained by merging the respective projections. RESULTS: Our experiments revealed that as platelets contact the substrate, a sequential and simultaneous rearrangement of actin filaments, myosin and tubulin molecules occurred and this was related to cell shape, as well as to movements of cytoplasmic granules. Treatment of platelets with cytoskeleton inhibitors, modified not only the target molecule but also other cytoskeletal components with consequent alterations in the studied platelet functions. INTERPRETATION AND CONCLUSIONS: During platelet adhesion to glass and granule displacement, a close spatial and functional relation between actin filaments, myosin molecules and microtubules was observed suggesting that these different cytoskeleton components interact in supporting the platelet functions here studied.     

肌纤形成调节因子1促进微丝修复减轻心肌细胞缺氧复氧损伤的研究

目的研究肌纤形成调节因子1(MR-1)通过促进心肌细胞微丝修复减轻心肌细胞缺氧/复氧损伤。方法原代培养的乳大鼠心肌细胞,随机分为正常对照组、模型组、过表达组、敲低组、过表达+模型组、敲低+模型组、转染对照组。采用RT-PCR检测MR-1、肌球蛋白调节型轻链2(MLC-2)mRNA表达,Western blot检测MR-1、纤维状肌动蛋白(F-actin)/球状肌动蛋白(G-actin)、MLC-2蛋白相对含量和磷酸化,荧光染色法检测F-actin。结果与模型组比较,过表达+模型组F-actin排列紊乱,其F-actin/G-actin比值升高22.4%(P<0.01),MLC-2mRNA和蛋白表达和磷酸化分别升高26.8%、24.8%和29.6%(P<0.01);而敲低+模型组F-actin排列紊乱,F-actin/G-actin比值降低10.1%(P<0.01),MLC-2mRNA和蛋白表达及磷酸化分别降低18.8%、55.9%和37.6%(P<0.01)。结论 MR-1通过激活MLC-2/F-actin途径修复微丝,减轻心肌细胞缺氧/复氧损伤。