腺苷对缺氧/复氧心肌细胞的保护作用

本研究旨在探讨腺苷 (adenosine ,ADO)对缺氧 /复氧 (hypoxia/reoxygenation ,H/R)心肌细胞的保护作用及其分子机制。将原代培养的新生大鼠心肌细胞分成H/R对照组和ADO (1 0 μmol/L)保护组。用倒置相差显微镜观察心肌细胞的生长状态。检测两组培养基质乳酸脱氢酶 (LDH)活性和心肌细胞Ca2 + 和丙二醛 (MDA)浓度。用ELISA法检测肿瘤坏死因子 (TNF α)的表达 ,并用凝胶电泳迁移率改变法 (EMSA)测定核因子 (NF κB)结合活性。所得结果如下 :(1)心肌细胞H/R培养后皱缩、变圆 ,伪足减少 ,ADO组心肌细胞的形态变化小于对照组 ;(2 )ADO减少缺氧和复氧期间心肌细胞LDH的漏出 (bothP <0 0 1) ;(3 )ADO降低缺氧和复氧期间心肌细胞内的Ca2 +浓度 (bothP <0 0 1) ;(4)ADO降低缺氧和复氧期间心肌细胞MDA浓度 (bothP <0 0 1) ;(5 )ADO抑制缺氧和复氧期间TNF α的表达 (bothP <0 0 1) ;(6)ADO抑制缺氧和复氧期间心肌细胞NF κB结合活性 (bothP <0 0 1)。以上结果提示 :(1)外源性ADO可减轻心肌细胞的H/R损伤 ;(2 )外源性ADO抑制H/R期间心肌细胞TNF α的表达 ;(3 )外源性ADO可能通过抑制心肌细胞NF κB结合活性下调TNF α的表达     

碱性成纤维细胞生长因子对乳鼠心肌细胞缺氧复氧损伤的影响

本实验在培养的乳鼠心肌细胞缺氧复氧损伤模型上观察碱性纤维细胞生长因子对Ａ／Ｒ损伤及蛋白激酶活性的影响,以探讨ｂＥＧＦ作为药物预处理心肌保护的可行性及其机理。结果表明,ｂＥＧＦ预处理呈浓度依赖地提高Ａ／Ｒ后心肌细胞存活率,减少细胞内ＡＴＰ消耗及胞浆乳酸脱氢酶漏出;ＰＫＣ抑制剂Ｈ７完全消除ｇＦＧＦ的上述保护作用;实验结果表明,ｂＦＧＦ可以直接激活心肌细胞ＰＫＣ,其激活时相的变化与缺氧预处理者相近,提示     

细胞粘附分子在中性粒细胞介导的缺氧／复氧心肌细胞损伤中的作用

目的观察缺氧/复氧对心肌细胞与中性粒细胞粘附效应的影响及细胞间粘附分子-1(intercellularadhensionmolecule-1,ICAM-1)和淋巴细胞功能相关抗原-1(lymphocytefunctionassociatedantigen-1,LFA-1)在中性粒细胞介导的心肌细胞损伤的作用。方法计数不同实验条件下与心肌细胞粘附的中性粒细胞;以及抗ICAM-1单抗和抗LFA-1单抗阻断后中性粒细胞粘附数的改变,检测心肌细胞乳酸脱氢酶释放量。结果中性粒细胞与缺氧/复氧心肌细胞粘附数较缺氧组和正常对照组显著增加(P＜0.01);心肌细胞释放LDH明显增高(P＜0．01),单纯缺氧组与正常对照组相比无显著差异(P＞0．05)。加入抗ICAM-1单抗和抗LFA-1单抗后,缺氧/复氧组与心肌细胞粘附的中性粒细胞数较正常对照组显著降低(P<0．01),心肌细胞释放LDH也明显下降(P＜0．01)。缺氧组与正常对照组相比则无显著差异(P＞0.05)。结论缺氧/复氧使心肌细胞与中性粒细胞粘附效应增加,心肌细胞损伤加重,ICAM-1和LFA-1参与这一过程。抗ICAM-1和抗LFA-1单抗可减轻中性粒细胞对缺氧/复氧心肌细胞的损伤。     

磷酸肌酸对豚鼠心肌细胞缺氧复氧损伤膜电位的影响

本实验观察了缺氧复氧对豚鼠心肌细胞膜电位的影响及磷酸肌酸的保护作用。结果表明缺氧５－２０ｍｉｎ时ＲＰ、ＡＰＡ减小,Ｖｍａｘ减慢,ＡＰＤ１０、ＡＰＤ５０、ＡＰＤ９０缩短。复氧５－２０ｍｉｎ后,ＲＰ、ＡＰＡ和Ｖｍａｘ进一步减少,ＡＰＤ１０、ＡＰＤ５０、ＡＰＤ９０明显缩短。在灌流液中加入磷酸肌酸保持其浓度在１０－６ｍｏｌ时,ＲＰ、ＡＰＡ显著增加,Ｖｍａｘ增快,ＡＰＤ１０、ＡＰＤ５０、ＡＰＤ９０明显延长。复氧２０ｍｉｎ后膜电位各项参数趋向恢复正常。此结果提示,磷酸肌酸对缺氧复氧的心肌具有显著的保护作用。     

金属硫蛋白对缺氧／复氧心肌细胞的保护作用

金属硫蛋白 (ＭＴ)是普遍存在各种生物体内、富含硫基的小分子蛋白 ,在缺血 /再灌注 (Ｉ/Ｒ)损伤中通过清除自由基减轻心肌细胞损伤 ,同时ＭＴ参与预缺血保护 (ＩＰＣ)。目前对ＭＴ在ＩＰＣ中的保护作用机制及功效仍无明确研究 ,本实验以培养的乳鼠心肌细胞缺氧 /复氧模拟缺血 /再灌注损伤 ,检测心肌细胞在ＩＰＣ处理后 2 4ｈＭＴ的含量、脂质过氧化程度及Ｎａ Ｋ ＡＴＰ酶、Ｃａ2 Ｍｇ2 ＡＴＰ酶的活力变化 ,锌诱导产生的ＭＴ作用 ,并比较ＭＴ与抗氧化剂谷胱甘肽 (ＧＳＨ)的保护作用 ,初步探讨ＭＴ的心肌保护作用机制及应用价值。1　…     

番茄红素对大鼠乳鼠心肌细胞缺氧复氧的保护作用以及其机制

目的:探讨番茄红素对心肌细胞缺氧复氧的保护作用以及其分子机制。方法:采用原代培养心肌细胞建立缺氧/复氧损伤模型,实验分8组:正常对照组,H/R组,H/R+番茄红素(1,2,4,8,16,32μmol/L)剂量组。观察各组细胞经H/R损伤后,细胞内天冬氨酸氨基转移酶(AST)、肌酸激酶(CK)、乳酸脱氢酶(LDH)、超氧化物歧化酶(SOD)活性和丙二醛(MDA)含量的变化情况,选择正常对照组,H/R组,最佳番茄红素剂量组做MTT分析细胞凋亡,Western检测TRL 4以及NF-κB的表达。结果:番茄红素(16,8,4,2μmol/L)剂量组可显著降低缺氧/复氧损伤心肌细胞内AST、CK、LDH释放量及MDA的生成,并能提高SOD活性。此外番茄红素可减少心肌细胞缺氧/复氧损伤后的心肌凋亡,减少TRL 4受体以及NF-κB的表达。结论:番茄红素具有抗缺氧/复氧损伤,保护心肌细胞的作用,其机制可能是通过抑制TRL 4通路来实现的。     

番茄红素对大鼠乳鼠心肌细胞缺氧复氧的保护作用以及其机制

目的：探讨番茄红素对心肌细胞缺氧复氧的保护作用以及其分子机制。方法：采用原代培养心肌细胞建立缺氧/复氧损伤模型,实验分8组：正常对照组,H/R组,H/R＋番茄红素（1,2,4,8,16,32μmol/L）剂量组。观察各组细胞经H/R损伤后,细胞内天冬氨酸氨基转移酶（AST）、肌酸激酶（CK）、乳酸脱氢酶（LDH）、超氧化物歧化酶（SOD）活性和丙二醛（MDA）含量的变化情况,选择正常对照组,H/R组,最佳番茄红素剂量组做MTT分析细胞凋亡,Western检测TRL 4以及NF-κB的表达。结果：番茄红素（16,8,4,2μmol/L）剂量组可显著降低缺氧/复氧损伤心肌细胞内AST、CK、LDH释放量及MDA的生成,并能提高SOD活性。此外番茄红素可减少心肌细胞缺氧/复氧损伤后的心肌凋亡,减少TRL 4受体以及NF-κB的表达。结论：番茄红素具有抗缺氧/复氧损伤,保护心肌细胞的作用,其机制可能是通过抑制TRL 4通路来实现的。     

缺氧复氧心肌细胞中HSP90对AKT表达的调控作用

研究新生大鼠缺氧复氧心肌细胞中热休克蛋白90(HSP90)的表达对AKT表达的影响,探讨HSP90在PI3K/AKT信号通路中的作用。方法:建立新生大鼠心肌细胞缺氧复氧模型,通过运用HSP90阻断剂Geldanamycin(GA),分另以MTT法检测心肌细胞的活力、透射电镜观察心肌细胞超微结构改变、Western印迹法分析大鼠心肌细胞中HSP90表达变化与总AKT蛋白的相关性。结果:缺氧复氧心肌细胞中HSP90和AKT表达量均有明显升高,阻断HSP90后,AKT表达量明显下降,此时心肌细胞活力明显下降,细胞超微结构受损明显。结论:AKT对缺氧复氧引起的心肌细胞损伤有内源性保护作用,此作用的发挥与HSP90和AKT的结合密切相关,HSP90对缺氧复氧中的心肌细胞AKT表达有显著影响。     

离体大鼠心肌细胞钠超负荷与缺氧—复氧损伤

本工作在离体成年大鼠心肌细胞缺氧-复氧模型上,观察到细胞无氧孵育时加入Na~ -K~ ATP酶抑制剂哇巴因,增加细胞内钠离子浓度,复氧孵育后造成了更严重的细胞损伤及钙超负荷,缺氧期末细胞内钠离子浓度与复氧后钙超负荷的程度呈显著正相关。复氧期给予Na~ -Ca~(2 )交换抑制剂Mn~(2 ),明显减轻了细胞的缺氧-复氧损伤,Mn~(2 )还显著抑制了无钠孵育引起的细胞损伤。结果提示:缺氧期细胞内钠超负荷是复氧时细胞内钙超负荷发生的条件,Na~ -Ca~(2 )交换是Ca~(2 )进入细胞的重要途径。     

缺氧预处理对乳鼠心肌细胞蛋白激酶C活性的影响

在培养的乳鼠心肌细胞缺氧／复氧模型上,观察了缺氧预处理的细胞保护作用及其对细胞蛋白激酶Ｃ活性和蛋白磷酸化的影响。结果表明,ＡＰＣ可减轻心肌细胞的Ｈ／Ｒ损伤;提高细胞存活率,减少细胞脂质过氧化产物生成及细胞内乳酸脱氢酶和蛋白质漏出。模拟ＡＰＣ的短暂缺氧显著激活ＰＫＣ,使心肌细胞内分子量为６６ｋＤ和３１ｋＤ的蛋白条带＾３２Ｐ掺入增加;ＰＫＣ抑制剂Ｈ７完全消除ＡＰＣ对心肌细胞的保护作用,并抑制了短暂缺氧     

Improved methods for automatic monitoring of contracting heart cells in culture

The present communication describes improved methods for isolating and plating beating heart cells from neonatal rats using collagenase and collagen-coated petri dishes. The amplitude and frequency of contraction are continuously and simultaneously measured under well defined conditions and during prolonged periods of time with a highly sensitive and thermostated instrument. Additions, e.g. drugs and toxic agents, are made through an accessory pump system that involves extensive dilution of the added compound with medium; aliquots of medium can be withdrawn for estimation of metabolites. The system described is reliable and relatively inexpensive and allows a more extensive use of isolated heart cells, especially in studies of heart functions where small changes in amplitude and frequency of beating during prolonged periods of time are important.     

缺氧/复氧早期对豚鼠心室肌持续性钠电流的影响

目的:研究心室肌细胞持续性钠电流(INa.P)在缺氧/复氧早期的变化,探讨其在此病理条件下的作用及意义。方法:运用全细胞膜片钳技术记录持续性钠电流,并观察其在缺氧-复氧模型下的变化。结果:①在0.5Hz,1Hz和2Hz的刺激频率下第1个和第8个刺激脉冲引起的INa.P电流密度差值分别为(0.021±0.014)pA/pF,(0.097±0.014)pA/pF和(0.133±0.024)pA/pF(P<0.01);②分别在-150~-80mV,阶跃10mV的钳制膜电位下去极化至-30mV,INa.P逐渐减小;③在缺氧条件下,INa.P电流密度增大,并随缺氧时间延长增大更显著;④在正常、缺氧15min和复氧5min时INa.P密度分别为(0.500±0.125)pA/pF,(1.294±0.321)pA/pF和(0.988±0.189)pA/pF(与对照比较P<0.01)。结论:以上特性提示INa.P在缺氧/复氧过程心律失常的产生及钙超载引起心肌损伤的机制中起重要作用。     

Studies on oxygen and volume restrictions in cultured cardiac cells I. A model for ischemia and anoxia with a new approach

Summary A novel incubation unit is described that is highly suitable for thorough studies of oxygen deprivation states. Its application with cultured heart cells is experimentally demonstrated. The release of enzymes, taken as a marker for cell damage, has clearly shown that restriction of the volume of extracellular medium combined with oxygen plus glucose deprivation caused greatest cellular damage. It may be considered as an experimental ischemia-like state. Furthermore, the onset of cellular damage followed a time table very much like that occurring in vivo under similar conditions, more so than any other previously described studies. A time lag between the release of cytoplasmic enzymes and lysosomal enzymes and other observations made in the present study suggests a sequential order of events in which the release of cytoplasmic enzymes occurs at a stage of reversible damage due to oxygen deprivation, whereas the release of lysosomal enzymes may point at irrepairable damage. Supported by grants from The Chief Scientist, Ministry of Health, State of Israel; The Ministry of Education and Sciences, State of Niedersachsen (FRG); and The Foundation for Heart Research from Mr. and Mrs. D. Vidal-Madjar, Paris, France. This study was done as partial fulfilment of Vemuri's Ph.D. thesis in biochemistry.     

低氧对CRF,AVP和NE刺激体外培养腺垂体细胞生成cAMP的影响

本文探讨了ＣＲＦ,ＡＶＰ及ＮＥ对体外培养大鼠腺垂体细胞生成ｃＡＭＰ的作用。ＣＲＦ刺激体外培养大鼠腺垂体细胞内ｃＡＭＰ的生成,且浓度与效应正相关。ＡＶＰ未引起细胞内ｃＡＭＰ差异性变化（Ｐ＞０．５）。ＮＥ使培养腺垂体细胞内ｃＡＭＰ水平降低。低氧使ＣＲＦ刺激ｃＡＭＰ生成的作用降低。而ＡＶＰ及ＮＥ可能是通过其它胞内信息通路。     

Reversibility of Cisternal Stack Formation During Hypoxic Hypoxia and Subsequent Reoxygenation in Cerebellar Purkinje Cells

Cisternal stacks are induced during hypoxia, which may be associated with intracellular Ca²⁺ regulation. Although neurons are divided internally in different compartments, little is known about regional differences in cisternal stack formation. We investigated the effects of hypoxic hypoxia and later reoxygenation on cisternal stack formation and other ultrastructual changes in the proximal dendrite, dendritic spine, and cell body of cerebellar Purkinje cells in rats. After brief hypoxic events, cisternal stacks appeared predominantly in the proximal dendrites and after longer hypoxic events in dendritic spines and cell body. Following reoxygenation, cisternal stacks disappeared first in the cell body, followed by the dendritic spines, then the proximal dendrites. These results showed that stack formation occurred at different degrees and time courses among the three regions, and the effect was reversible, which suggests that these compartments are differentially sensitive to hypoxia.     

组胺对培养新生大鼠心肌细胞自发性搏动及动作电位的影响

以培养大鼠乳鼠心肌细胞为模型,观察组胺对培养心肌细胞自发性搏动频率及动作电位的影响。结果表明：组胺０．１－１０ｕｍｏｌ／Ｌ可以引起剂量依赖性的心肌细胞搏动频率的增快,而且这种效应呈时间依赖性。组胺１０ｕｍｏｌ／Ｌ可以使心肌细胞动作电位的幅度（ＡＰＡ）,最大上升速率（Ｖｍａｘ）及超射（ＯＳ）明显增加,动作电位持续时间（ＡＰＤ５０和ＡＰＤ９０）明显延长,窦性周长（ＳＣＬ）明显缩短。以上结果提示,组胺致     

间歇性低氧处理大鼠心肌的抗心律失常与抗氧化效应

利用结扎在体大鼠冠脉方法研究不同时间间歇性低氧处理对血、再灌注心律失常以及心肌超氧化物歧化酶（ＳＯＤ）、丙二醛（ＭＤＡ）的影响,并与连续性低氧相比较。实验结果如下：⑴间歇性低氧（ｉｎｔｅｒｍｉｔｔｅｎｔ ｈｙｐｏｘｉａ ｅｘｐｏｓｕｒｅ）２８ｄ（ＩＨ２８）、４２ｄ（ＩＨ４２）、间歇性低氧２８ｄ后１周（ＰＩＨ２８－２Ｗ）和连续性低氧（ｃｏｍｔｉｎｕｅｄ ｈｙｐｏｘｉａ ｅｘｐｏｓｕｒｅ）２８ｄ（ＣＨ     

Biological Efficiency of AMP Deaminase Inhibitor: 3-[2-(3-CARBOXY-4-BROMO-5,6,7,8-Tetrahydronaphthyl)Ethyl]-3,6,7,8-Tetrahydroimidazo[4,5]-[1,3]Diazepin-8-OL

AMP deaminase could be a potential target for treatment of heart disease but experimental evaluation of this concept is difficult due to limited availability of inhibitors with proven efficiency in biological systems. This study evaluated the effect of 3-[2-(3-carboxy-4-bromo-5,6,7,8-tetrahydronaphthyl)ethyl]-3,6,7,8-tetrahydroimidazo [4,5-d][1,3]diazepin-8-ol, an AMP deaminase inhibitor (AMPDI) on the pathways of nucleotide metabolism in perfused rat heart. We show that AMPDI at 0.3 mM concentration effectively inhibits AMP deaminase in this experimental model.     

线粒体钙单向转运体在心肌低氧/复氧损伤中的作用

目的:观察线粒体钙单向转运体在心肌低氧/复氧损伤中的作用并探讨其机制。方法:应用Langendorff大鼠心脏灌流模型,低氧/复氧(H/R)采用冠脉前降支结扎30 min、复灌120 min的方法。用生物信号采集系统记录左室发展压(LVDP)、左室压最大上升/下降速率(±dP/dtmax)、左室舒张末压(LVEDP);分光光度法分别检测冠脉流出液中乳酸脱氢酶(LDH)的含量和线粒体活性氧(ROS);TTC染色法检测心肌梗死面积。结果:与单纯低氧/复氧组相比,复氧起始给予线粒体钙单向转运体抑制剂钌红(5μmol/L)明显改善左心室各项功能指标,减小心肌梗死面积,降低线粒体ROS和冠脉流出液中LDH含量;而在复氧期起始给予线粒体钙单向转运体激动剂精胺(20μmol/L),显著升高了线粒体ROS活性,冠脉流出液中LDH含量在复氧5 min、20 min、30 min时显著增多,左心室各项功能指标与心肌梗死面积与单纯低氧/复氧组相比无显著差异。ROS清除剂MPG(1 mmol/L)与精胺联合应用则取消了精胺的作用。结论:抑制线粒体钙单向转运体可能通过减少线粒体ROS的生成减轻心脏低氧/复氧损伤。     

Insulin induces changes in the subcellular distribution of actin and 5′-nucleotidase

The present study utilized a cultured adult myocardial cell model to examine the arachidonic acid metabolism under different cell-damaging and normoxic conditions. Cell injury was caused by short-time hypoxia, calcium ionophore A 23187-triggered cell-damage under hypoxia and cell disruption by freezing and thawing. The current study demonstrates that under the cell-damaging conditions cultured adult heart myocytes resemble myocardial cells under normoxic conditions in metabolizing arachidonic acid into triacylglycerols and phospholipids as the major route (a), in formation of ETYA-inhibitable indomethacin-resistant lipid metabolites in minor amounts (b) and in being independent of calcium overload in the metabolic pathways of arachidonic acid metabolism (c). The ETYA-inhibitable components were resolved by HPLC. There was no evidence in formation of lipoxygenase products. The results were supported by negative hybridisation experiments of the total mRNA isolated from adult myocardial cells with a cDNA probe of a red-cell-specific lipoxygenase mRNA. We conclude from these observations that cell injury does not result in expression of lipoxygenase activities in heart myocytes.Abbreviations HETE Hydroxyeicosatetraenoic acid - DiHETE Dihydroxyeicosatetraenoic acid - ETYA 5.8.11.14-Eicosatetraynoic acid - TLC Thin-Layer Chromatography - NP-HPLC Normal Phase-High Performance Liquid Chromatography - RBC Red Blood Cell - LOX Lipoxygenase