顿抑心肌中腺嘌呤核苷酸转运体基因的动态表达

目的:通过观察大鼠心肌缺血再灌注模型中腺嘌呤核苷酸转运体(adeninenucleotidetranslocator,ANT)的动态表达情况,初步探讨顿抑心肌中ANT各单体的表达变化对心肌能量代谢的影响及其与心肌细胞凋亡的联系。方法:实验于2003-11/2004-04在解放军南京军区南京总医院心脏内科实验室和动物实验中心完成。将60只雄性SD大鼠随机分为对照组(n=8)和手术组(n=52)。将手术组大鼠冠状动脉左前降支结扎闭塞20min,然后剪断结扎线再灌注。按缺血后再灌注4h、1,3,7d4个时相分4组进行观察(n=13)。用反转录-聚合酶链反应计算机凝胶成像分析系统测定心肌中ANT1,ANT2mRNA相对含量。对照组大鼠不干预。结果:60只大鼠进入结果分析。①ANT1mRNA含量在缺血后再灌注4h达高峰(P<0.05)。在再灌注1d开始降低,再灌注3d时明显下降(P<0.05),逐渐恢复到对照水平(P>0.05),再灌注7d的结果与再灌注3d相似。②ANT2mRNA含量在再灌注4h时无明显增加,在再灌注1d开始升高并达峰值(P<0.01),从再灌注3d开始明显下降(P<0.01),并恢复到对照水平(P>0.05)。再灌注7d结果与再灌注3d相似。结论:①顿抑心肌中ANT的含量增加,顿抑心肌中能量短缺的现象可在缺血后再灌注3d内得以纠正。②心肌缺血可以导致细胞凋亡,心肌细胞凋亡主要发生在缺血再灌注早期(24h内),而且在体内存在时间短暂(缺血后再灌注3d内恢复)。     

顿抑心肌中葡萄糖转运体基因的动态表达

目的:通过观察大鼠心肌缺血再灌注模型中葡萄糖转运体(glucose transporters,GLUT)的动态表达变化,初步探讨GLUT1 mRNA和GLUT4 mRNA的表达变化对顿抑心肌能量代谢的影响.方法:将60只雄性SD大鼠随机分为对照组和手术组.手术组将大鼠冠状动脉左前降支完全闭塞20 min,然后剪断结扎线再灌注.按缺血后再灌注4 h、1 d、3 d、7 d 4个时相进行观察,用RT-PCR计算机凝胶成像分析系统检测心肌GLUT1 mRNA和GLUT4 mRNA 的相对含量.结果:心肌缺血20 min后,再灌注4 h GLUT1 mRNA和GLUT4 mRNA的相对含量均达峰值,两者与对照组相比差异均有极显著性(0.666±0.003 vs 0.509±0.002,1.190±0.007 vs 0.937±0.034,均P＜0.01).GLUT1mRNA含量在再灌注7 d后仍明显升高(P＜0.01),GLUT4 mRNA含量从再灌注3 d后开始下降,与对照组无明显差异(P＞0.05).结论:顿抑心肌中GLUT1 mRNA和GLUT4 mRNA的表达增加,促进了缺血心肌对葡萄糖的利用,保护缺血心肌,有利于缺血再灌注后心肌功能的恢复.     

大鼠心肌缺血再灌注损伤与心肌肽素的保护作用

目的：探讨心肌肽素对大鼠心肌缺血再灌注损伤的保护作用及作用途径。方法：实验于2004-04/06在阜外心血管病医院麻醉研究室完成。选择健康雄性SD大鼠24只，随机分为3组，每组8只，分别为假手术组、缺血再灌注组、心肌肽素用药组。大鼠冠状动脉左前降支结扎30min造成心肌缺血模型，复灌24h造成心肌缺血再灌注损伤模型。①再灌注结束后大鼠血清肌酸激酶同工酶、乳酸脱氢酶水平采用全自动生化仪检测。②检测大鼠心肌细胞凋亡指数应用原位末端标记法。③心肌肌动蛋白表达采用特异性免疫组化二步法检测。④大鼠心肌病理学改变光镜下半定量评分以10倍物镜下，随机选取若干视野观察整个左室心肌切面，从心肌变性坏死、出血、间质水肿和中性粒细胞浸润四方面观察心肌病变分布情况并计分，计算每种病变的视野比例(有心肌病变的视野数/镜下观察的总视野数)，正常心肌计0分；病变灶性，病变视野比例≤1／4计1分；病变视野比例为1／4～1／2计2分；病变弥散性，病变视野比例≥1／2计3分。每方面的得分求和即为最后计分。⑤大鼠心肌病理学电镜下半定量评分：从心肌纤维、线粒体、核染色质、细胞水肿和小血管内皮五方面观察其超微结构改变并计分，结构正常为0分；轻度改变且病变灶性，小于全部视野面积的1／4为1分；中度改变，病变占全部视野面积的1／4～1／2计2分；重度改变且病变弥散性，大于全部视野面积的1／2为3分。每方面的得分求和即为最后计分。结果：进入结果分析大鼠24只，每组8只。①大鼠血清肌酸激酶同工酶、乳酸脱氢酶水平、心肌细胞凋亡指数：心肌肽素用药组明显低于缺血再灌注组[(5．59&;#177;0．52)μ kat/L，(1．06&;#177;0．09)μkat／L，(4．34&;#177;1．15)％；(10．52&;#177;0.32)μ kat/L，(1．97&;#177;0.21)μ kat／L，(11．16&;#177;1．09)％，P&;lt;0．05]，缺血再灌注组、心肌肽素用药组明显高于假手术组[(2．58&;#177;0．32)μ kat／L，(0．57&;#177;0．15)μ kat／L，（0．85&;#177;0．42)％，P&;lt;0．05]。②大鼠心肌肌动蛋白含量和电镜、光镜评分组间差异性与血清酶学检测心肌细胞凋亡指数组间差异相同。结论：心肌肽素通过抑制心肌细胞凋亡，减轻心肌细胞结构损伤发挥对大鼠心肌缺血再灌注损伤的保护作用。     

腺嘌呤核甘酸转运子-1在大鼠压力超负荷致心肌重塑中的表达

目的：探讨心肌线粒体腺嘌呤核甘酸转运子-1（adenine nucleotide translocator-1,ANT1）对压力超负荷致心肌重塑的影响.方法：利用腹主动脉缩窄法建立大鼠压力超负荷模型,假手术组大鼠3只作为对照组.观察时间点为术后1,2,4,7,14,21和30天.ANT1 mRNA含量的测定采用RT-PCR法,利用TUNEL法检测心肌细胞的凋亡.心肌纤维化的改变采用心肌胶原形态定量分析.结果：①与对照组比较,缩窄后4天ANT1 mRNA的含量上调,并于第7天达到峰值,术后14天其含量回落至对照组水平,并保持至30天.②手术组心肌细胞凋亡在术后1天即升高,在4天时进入高峰期并持续至7天,其后低水平持续存在,直至实验结束.而对照组未发现凋亡的存在.③与对照组比较,心肌胶原容积分数于术后14天方明显增加,并持续增高.结论：ANT1参与了压力超负荷致心肌重塑早期心肌细胞凋亡的调节,并可能影响了这一过程的发生和发展.     

大鼠心肌缺血再灌注中细胞凋亡及Bcl-2、C-myc的表达研究

目的观察心肌缺血再灌注时心肌细胞凋亡现象,研究Bcl-2、C-myc蛋白的表达情况。方法采用末端标记技术(TUNEL)标记凋亡细胞,免疫组织化学法检测Bcl-2、C-myc蛋白的表达。结果在持续缺血和缺血再灌注大鼠心肌中,TUNEL法检测到大量的阳性细胞。随着再灌注时间的延长,大鼠心肌细胞凋亡数逐渐增多。持续缺血4.5 h组的心肌细胞凋亡数明显高于缺血30min再灌注4 h组的凋亡心肌细胞数。免疫组织化学法检测发现,与假手术组比较,持续缺血4.5 h组和缺血30min再灌注4 h组Bcl-2、C-myc蛋白的表达都增加,缺血再灌注组中Bcl-2蛋白表达明显上调,C-myc蛋白表达明显下调。结论心肌缺血再灌注中存在细胞凋亡现象,且心肌细胞凋亡与Bcl-2、C-myc蛋白的表达密切相关。     

组织因子反义寡脱氧核苷酸防治心肌缺血再灌注损伤的实验研究

目的:研究组织因子(TF)反义寡脱氧核苷酸(AS/TF)对大鼠心肌缺血再灌注损伤的作用。方法:设计针对大鼠TF的AS/TF、正义寡脱氧核苷酸(S/TF)和错配寡脱氧核苷酸(Sc/TF)。50只雄性Wistar大鼠随机分为假手术组(Sham组)、缺血再灌注损伤组(I/R组)、反义寡脱氧核苷酸防治组(AS/TF组)、正义寡脱氧核苷酸防治组(S/TF组)和错配寡脱氧核苷酸防治组(Sc/TF组),每组10只。Sham组和I/R组大鼠经静脉注入生理盐水,AS/TF组、S/TF组和Sc/TF组大鼠分别注入AS/TF、S/TF和Sc/TF。于注射后10h麻醉大鼠,开胸暴露心脏,于冠状动脉左前降支中1/2处穿线,Sham组只穿线不结扎,其余4组均结扎造成心肌缺血,90min后解除结扎,再灌注1h。用ELISA检测大鼠血清心肌肌钙蛋白I(cTnI)、血浆凝血酶鄄抗凝血酶复合物(TAT)和血小板P鄄选择素的含量。Northern印迹杂交检测大鼠缺血区心肌组织中TF基因的转录,HE染色观察缺血区心肌组织的病理变化。结果:大鼠心肌缺血再灌注后,血清cTnI、血浆TAT和P鄄选择素含量明显上升(P<0.001),AS/TF组的上升幅度低于I/R组、S/TF组和Sc/TF组(P<0.05)。Northern印迹杂交显示大鼠缺血区心肌组织TF基因的转录明显增强,AS/TF组TFmRNA的转录弱于I/R组、S/TF组和Sc/TF组。HE染色可见大鼠缺血区心肌组织有灶性坏死、心肌纤维     

川芎嗪对实验性缺血-再灌注损伤心肌能量代谢的干预

目的　探讨川芎嗪对心肌缺血—再灌注损伤 (MIRI)时心肌细胞能量代谢的影响。方法　实验兔 30只 ,随机分为正常对照组 (A组 )、MIRI组 (B组 )和MIRI+川芎嗪治疗组 (C组 )。复制MIRI模型 ,分别观察心肌组织内三磷酸腺苷 (ATP)、二磷酸腺苷 (ADP)、一磷酸腺苷 (AMP)含量、总腺苷酸量 (TAN)、能荷 (EC)及心肌超微结构的改变。结果　C组与B组比较 ,心肌组织内ATP、ADP、TAN含量及EC均明显增高〔ATP :C组为 (5 0 5± 1 34) μmol/g(湿重 ) ,B组为 (2 86± 1 2 2 ) μmol/g(湿重 ) ,P <0 0 1;ADP :C组为 (1 6 7± 0 4 0 ) μmol/g(湿重 ) ,B组为 (1 12± 0 2 5 ) μmol/g(湿重 ) ,P <0 0 1;TAN :C组为 (8 4 2± 1 90 ) μmol/g(湿重 ) ,B组为 (5 83± 1 5 5 ) μmol/g(湿重 ) ,P <0 0 1;EC :C组为 0 70± 0 0 3,B组为 0 5 8± 0 0 7,P <0 0 1〕 ;且与A组比较 ,ADP、AMP、TAN无明显差异〔ADP :A组为 (2 0 4± 0 5 3) μmol/g(湿重 ) ,P >0 0 5 ;AMP :A组为 (1 4 5± 0 2 9) μmol/g (湿重 ) ,P >0 0 5 ;TAN :A组为 (10 2 6± 2 0 5 ) μmol/g(湿重 ) ,P>0 0 5〕 ;心肌超微结构的异常改变显著减轻。结论　川芎嗪可改善缺血—再灌注损伤心肌的能量代谢。     

大鼠心肌缺血再灌注损伤与心肌肽素的保护作用

目的:探讨心肌肽素对大鼠心肌缺血再灌注损伤的保护作用及作用途径。方法:实验于2004-04/06在阜外心血管病医院麻醉研究室完成。选择健康雄性SD大鼠24只,随机分为3组,每组8只,分别为假手术组、缺血再灌注组、心肌肽素用药组。大鼠冠状动脉左前降支结扎30min造成心肌缺血模型,复灌24h造成心肌缺血再灌注损伤模型。①再灌注结束后大鼠血清肌酸激酶同工酶、乳酸脱氢酶水平采用全自动生化仪检测。②检测大鼠心肌细胞凋亡指数应用原位末端标记法。③心肌肌动蛋白表达采用特异性免疫组化二步法检测。④大鼠心肌病理学改变光镜下半定量评分以10倍物镜下,随机选取若干视野观察整个左室心肌切面,从心肌变性坏死、出血、间质水肿和中性粒细胞浸润四方面观察心肌病变分布情况并计分,计算每种病变的视野比例(有心肌病变的视野数/镜下观察的总视野数),正常心肌计0分;病变灶性,病变视野比例≦1/4计1分;病变视野比例为1/4～1/2计2分;病变弥散性,病变视野比例≧1/2计3分。每方面的得分求和即为最后计分。⑤大鼠心肌病理学电镜下半定量评分:从心肌纤维、线粒体、核染色质、细胞水肿和小血管内皮五方面观察其超微结构改变并计分,结构正常为0分;轻度改变且病变灶性,小于全部视野面积的1/4为1分;中度改变,病变占全部视野面积的1/4～1/2计2分;重度改变且病变弥散性,大于全部视野面积的1/2为3分。每方面的得分求和即为最后计分。结果:进入结果分析大鼠24只,每组8只。①大鼠血清肌酸激酶同工酶、乳酸脱氢酶水平、心肌细胞凋亡指数:心肌肽素用药组明显低于缺血再灌注组[(5.59±0.52)μkat/L,(1.06±0.09)μkat/L,(4.34±1.15)%;(10.52±0.32)μkat/L,(1.97±0.21)μkat/L,(11.16±1.09)%,P<0.05],缺血再灌注组、心肌肽素用药组明显高于假手术组[(2.58±0.32)μkat/L,(0.57±0.15)μkat/L,(0.85±0.42)%,P<0.05]。②大鼠心肌肌动蛋白含量和电镜、光镜评分组间差异性与血清酶学检测心肌细胞凋亡指数组间差异相同。结论:心肌肽素通过抑制心肌细胞凋亡,减轻心肌细胞结构损伤发挥对大鼠心肌缺血再灌注损伤的保护作用。     

老年大鼠心肌再灌注时ICAM-1表达变化规律及艾司洛尔的影响

目的：探讨细胞间粘附分子－1（ICAM－1）表达与老年大鼠心肌缺血再灌注损伤（IRI）的关系，并观察艾司洛尔（ES）对IRI的影响。方法：大鼠116只，分设缺血再灌注（IR）组，IR＋ES组和假手术对照组，并分设缺血1h，再灌注3、6、12、24h时相点，取缺血心肌用原位杂交和免疫组织化学方法检测ICAM－1 mRNA及其蛋白质表达水平，用酶法测定中性粒细胞（PMNs)浸润数，硫代巴比妥酸比色法测定心肌组织丙二醛（MDA），用黄嘌呤过氧化物酶法测定过氧化物歧化酶（SOD）活性，TTC染色法测定梗死范围。结果：心肌IR时，ICAM－1表达、MDA含量、PMNs浸润数均明显增高，SOD活性明显降低；ICAM－1蛋白表达水平、PMNs浸润与心肌梗死范围呈显著正相关，但ICAM－1 mRNA、MDA、SOD与梗死范围无明显相关性。IR＋ES组上述指标于再灌注时虽也明显增高，但比IR组明显减轻。结论：心肌IR时，ICAM－1参与介导了PMNs对组织细胞的粘附、浸润和IRI的发生、发展；ES可通过抑制ICAM－1的表达而产生心肌保护作用。     

Platelet adenine nucleotide levels during room-temperature storage of platelet concentrates

Platelet adenine nucleotide levels were measured in freshly prepared platelet concentrates and daily during storage at room temperature. Marked depletions in platelet adenosine triphosphate (ATP) and adenosine diphosphate (ADP) levels were associated with decrease in both plasma glucose levels and poststorage pH. In those concentrates with only moderate glucose consumption, platelet ATP and ADP levels were well preserved and the plasma pH remained above 6.0. The rate of glucose utilization and poststorage pH were greatly influenced by the concentration of contaminating leukocytes. These studies indicate that the rate of exogenous glucose consumption is an important factor affecting platelet adenine nucleotide levels during room temperature storage of platelet concentrates.     

Allopurinol enhanced adenine nucleotide repletion after myocardial ischemia in the isolated rat heart.

Allopurinol, a competitive inhibitor of xanthine oxidase, has been shown to have a protective effect on ischemic myocardium, but its mechanism of action remains controversial. We used an isolated rat heart preparation to test the hypothesis that allopurinol could restore adenosine triphosphate (ATP) levels and improve the recovery of left ventricular function after global myocardial ischemia. Hearts were equilibrated for 30 min, subjected to 10 min of global, normothermic (37 degrees C) ischemia, and reperfused for 15, 30, and 60 min. Hearts treated with allopurinol (100 microM) exhibited greater ATP levels and improved function during reperfusion than did untreated control hearts. Hearts treated with hypoxanthine (100 microM), the substrate for xanthine oxidase, also showed increased ATP and functional recovery compared with controls. These results suggest that allopurinol may protect the globally ischemic myocardium by enhancing the salvage of hypoxanthine for reincorporation into adenine nucleotides.     

心肌顿抑及运动的影响

背景:随着体育科研领域研究的深入,运动对心脏的影响日益受到体育科研人员的关注和重视.目的:系统综述心肌顿抑的研究历程、机制以及运动对顿抑心肌的影响.方法:由第一作者检索PubMed数据库及维普数据库(1980-01/2010-01)的相关文章.英文检索词为"myocardial stunning;exercises;ischemia-reperfusion",中文检索词为"心肌顿抑;运动;缺血再灌注".计算机初步检索到83篇文献按纳入和排除标准筛选出36篇文献,从心肌顿抑的定义、特点、分类、机制以及运动对心肌顿抑影响等几方面出发进行归纳总结.结果与结论:心肌顿抑在临床医学研究中越来越深入,心肌顿抑的定义、特点以及分类都很明确,并且也开始从分子生物学层面探索心肌顿抑的可能机制,然而剧烈运动持续时间过长,可引起运动员心肌灌流不足,导致心功降低和心肌损伤,由此可见,某种条件下,运动员可能出现心肌顿抑现象.     

Acidosis during early reperfusion prevents myocardial stunning in perfused ferret hearts.

Cellular calcium overload figures prominently in the pathogenesis of the contractile dysfunction observed after brief periods of ischemia (myocardial stunning). Because acidosis is known to antagonize Ca influx and the intracellular binding of Ca, we reasoned that acidosis during reperfusion might prevent Ca overload and ameliorate functional recovery. We measured developed pressure (DP) and 31P-nuclear magnetic resonance spectra in 26 isovolumic Langendorff-perfused ferret hearts. After 15 min of global ischemia, hearts were reperfused either with normal solution (2 mM [Ca]o, Hepes-buffered, pH 7.4 bubbled with 100% O2; n = 6) or with acidic solutions (pH 6.6 during 0-3 min, pH 7.0 during 4-6 min) before returning to the normal perfusate (n = 7). Ventricular function after 30 min of reperfusion was much greater in the acidic group (105 +/- 5 mmHg at 2 mM [Ca]o) than in the unmodified reperfusion group (79 +/- 7 mmHg, P less than 0.001); similar differences in DP were found over a broad range of [Ca]o (0.5-5 mM, P less than 0.001) and during maximal Ca2+ activation (P less than 0.001). Intramyocardial pH (pHi) was lower in the acidic group than in the unmodified group during early reperfusion, but not at steady state. Phosphate compounds were comparable in both groups. To clarify whether the protective effect of acidosis is due to intracellular or extracellular pH, we produced selective intracellular acidosis during early reperfusion by exposure to 10 mM NH4Cl for 6 min just before ischemia (n = 6). For the first 12 min of reperfusion with NH4Cl-free solution (pH = 7.4), pHi was decreased relative to the unmodified group. Recovery of DP was practically complete, and maximal Ca2+-activated pressure was comparable to that in a nonischemic control group (n = 5). These results indicate that transient intracellular acidosis can prevent myocardial stunning, presumably owing to a reduction of Ca influx into cells and/or competition of H+ for intracellular Ca2+ binding sites during early reperfusion.     

Relationship between myocardial adenine nucleotide catabolism and tissue blood flow rate in experimental ischaemia

A method was developed for tissue preservation and evaluation of the adenine nucleotide metabolism in small samples of myocardium after 45 min of ischaemia. Ischaemia was produced by coronary artery occlusion in anaesthetized cats. Adenine nucleotides and their metabolites were measured by isocratic liquid chromatographic systems which allow quantitative analysis of the nucleotides and their metabolites inosine, hypoxanthine and xanthine in biopsies of 5-20 mg tissue. Regional myocardial blood flow was measured in the tissue surrounding the biopsies by the distribution of 15 micron radiolabelled microspheres. In central ischaemic regions the ATP level was approximately 1 mumol/g wet weight, whereas in normally perfused myocardium the ATP level was approximately 5 mumol/g tissue. In tissue with intermediate flow values, intermediate ATP levels were found. Energy charge, which summarizes all adenine nucleotide concentrations, was reduced from 0.88 to 0.50, and the molar concentrations of inosine, hypoxanthine and xanthine increased in ischaemic tissue. We conclude that this method provides reliable characterization of the local cellular energy status in cat hearts with ischaemic regions.     

Pathways of adenine nucleotide catabolism in erythrocytes.

The exact pathway whereby the initial catabolism of the adenine nucleotides proceeds from AMP and the possibility of a recycling of adenosine were investigated in human erythrocytes. Adenine nucleotide catabolism, reflected by the production of hypoxanthine, is very slow under physiologic conditions and can be greatly increased by suppression of glucose or alkalinization of the medium. Experiments with inhibitors of adenosine deaminase and adenosine kinase demonstrated that under physiologic conditions the initial catabolism of AMP proceeds by way of a deamination of AMP, followed by dephosphorylation of inosine monophosphate, and that no recycling occurs between AMP and adenosine. Under glucose deprivation, approximately 75% of the 20-fold increase of the catabolism of the adenine nucleotides proceeded by way of a dephosphorylation of AMP followed by deamination of adenosine, and a small recycling of this nucleoside could be evidenced. Inhibition of adenosine transport showed that the dephosphorylation of AMP occurred intracellularly. When the incubation medium was alkalinized in the presence of glucose, the 15-fold increase in the conversion of AMP to hypoxanthine proceeded exclusively by way of AMP deaminase but a small recycling of adenosine could also be evidenced. The threefold elevation of intraerythrocytic inorganic phosphate (Pi) during glucose deprivation and its 50% decrease during alkalinization as well as experiments in which extracellular Pi was modified, indicate that the dephosphorylation of red blood cell AMP is mainly responsive to variations of AMP, whereas its deamination is more sensitive to Pi.