培养心肌细胞内的SDH图象分析——大豆磷脂脂质体对缺血再灌心肌膜损伤的影响

琥珀酸脱氢酶(SDH)的活性在判断心肌缺血,再灌性损伤中起重要作用。本文通过IBAS图象分析系统对缺血再灌的培养心肌细胞内的SDH定量评估,以求探讨大豆磷脂脂质体对心肌缺血再灌性损伤的保护作用。结果表明,在缺血及再灌组的心肌细胞内出现较强的SDH损伤性反应,大豆磷脂脂质体能明显减轻这种反应,提示大豆磷脂脂质体对缺血及缺血/再灌心肌具有明显的保护作用。     

大鼠心肌整体缺血及离体再灌注致生物膜的损伤作用

目的和方法：利用整体大鼠异丙肾上腺素损伤（ISO）和离体大鼠全心停灌／再灌（I／R）两种模型,观察了心肌缺血和缺血／再灌注对心肌生物膜－线粒体膜及肌纤维膜损伤的影响。结果：ISO（5mg/kg,皮下注射）和I／R（20min/20min)可导致大鼠心脏生物膜产生严重损伤,表现为心肌线粒体脂质过氧化产物明显增加,线粒体磷脂酶A2（PLA2）激活,从而导致线粒体膜磷脂（PL）含量减少,磷脂分解产物游离脂肪酸（FFA）增加,膜脂流动性（LFU）降低,线粒体Ca^2 -ATPase及肌纤维膜Na^ ,K^ -ATPase活性降低,线粒体呼吸功能降低、呼吸链氧化磷酸化解偶联,高能磷酸化合物生成减少。结论：整体ISO和离体I／R可导致大鼠心肌线粒体、肌纤维膜结构和功能损伤。     

膜磷脂代谢与心肌缺血再灌注损伤

膜磷脂是维持细胞结构与功能的重要成份。心肌缺血—再灌注后导致膜磷脂降解,其含量明显减少,这是再灌注损伤的重要发病环节。用药物阻止膜磷脂降解可预防缺血心肌的再灌注损伤。     

磷脂模型膜的时间分辨纳秒荧光光谱的研究

叙述了用时间分辨率纳秒荧光技术研究大豆磷脂或合成磷脂模型膜（脂质体）的物理状态变化与荧光寿命以及时间分辨各向异性测量参数变化相关性的实验结果。用荧光探剂ＭＣ５４０标记模型膜的结果表明,荧光寿命（τ）的变化与脂质／探剂比例、磷脂组成、磷脂的极性头部、脂肪酸酰链长度等有关。用ＤＰＨ（１,６－ｄｉｐｈｅｎｙｌ－１,３,５－ｈｅｘａｔｒｉｅｎｅ）标记磷脂模型膜的时间分辨各向异性分析结果表明,序参数（Ｓ）、     

一氧化氮在心肌缺血再灌注损伤中的作用

目的：观察一氧化氮（NO）对相对缺血再灌注心肌损伤的保护作用。方法：高频弱电流刺激法建立离体心肌相对缺血再灌注模型,设非缺血组和相对缺血组,相对缺血组包括对照、L－精氨酸（L－ARG）、硝基－L－精氨酸甲酯（L－NAME）三组。测定缺血前和再灌注时心功能变化、NO含量和乳酸脱氢酶同工酶－1（LD－1）活性。结果：L－ARG可明显促进再灌注期间NO合成,抑制D－1活性升高。再灌注40min时,L－ARG组心肌功能恢复程度明显高于对照组和L－NAME组（P＜0.05）,L－NAME使心肌NO含量降低（P＜0.05）,LD－1活性升高（P＜0.05）,心功能恢复程度最低。结论：NO可明显减轻心肌缺血再灌注时的细胞损伤,促进心功能的恢复。     

粉防己碱对大鼠心肌缺血再灌注时心肌ATP酶活性的影响

实验旨在观察在体大鼠短暂缺血后心肌膜ＡＴＰ酶活性的变化及粉防己碱（Ｔｅｔ）的作用。分离缺血１５ｍｉｎ、再灌注２ｈ后及在缺血再灌注前给Ｔｅｔ的大鼠心肌粗制质膜和内质网,测定质膜Ｎａ＋－Ｋ＋－ＡＴＰ酶和内质网Ｃａ２＋－ＡＴＰ酶活性。结果表明,心肌缺血１５ｍｉｎ后二酶活性均明显降低,分别为假结扎组的６３．６％和７２．６％（Ｐ＜０．０１）,再灌注后Ｎａ＋－Ｋ＋－ＡＴＰ酶活性有所恢复,再灌注３０ｍｉｎ时为假结扎组的７２．１％（Ｐ＜０．０１）,而Ｃａ２＋－ＡＴＰ酶活性则进一步下降,再灌注３０ｍｉｎ时为假结扎组的５０．４％（Ｐ＜０．０１）,再灌注后２ｈ二酶活性分别升高至假结扎组的８０．９％和６５．３％（Ｐ＜０．０１）。在缺血前２０ｍｉｎ分别给予Ｔｅｔ６４．２和９６．３μｍｏｌ／ｋｇ及硝苯啶（０．２３μｍｏｌ／ｋｇ）,能明显减少内质网Ｃａ２＋－ＡＴＰ酶活性的降低。结果提示心肌膜ＡＴＰ酶活性的降低可能参与了短暂心肌缺血所致再灌注损伤的发生机制,Ｔｅｔ可减少缺血和／或再灌注时内质网Ｃａ２＋－ＡＴＰ酶活性降低。     

生物膜组分对膜功能和膜脂相变的调控——Ⅲ.磷脂脂质体对玉米根端线粒体ATP酶活力的影响

大豆粗磷脂经硅酸柱层析分离得到端基脂肪酸链相似的PC和PE+PG两种混合物。经超声制备成PC脂质体和PE+PG脂质体,添加于玉米根端线粒体,线粒体ATP酶活化能的折点温度由15.5℃分别降低为13℃和10.7℃,表明脂质体能与线粒体膜相结合,磷脂极性头基对ATP酶活化能的折点温度具有明显的作用。     

川芎嗪对缺血心肌的保护及抗氧化作用的实验研究

研究川芎嗪的心肌保护及抗氧化作用机制。在心脏停跳液中加入川芎嗪后对离全兔心脏进行缺血再灌注,然后观察心肌线粒体内丙二醛（ＭＤＡ）含量、超氧化物岐化酶（ＳＯＤ）活性及心肌组织超微结构损伤程度。发现含有川芎嗪的停跳液组丙二醛（ＭＤＡ）含量显著降低,超氧化物岐化酶（ＳＯＤ）活显著升高,观察心肌组织超微结构损伤程度发现较轻,因此,川芎嗪对缺血心肌具有良好的保护脑抗氧化作用。     

异丙酚对离体大鼠心肌缺血、再灌注损伤后细胞凋亡及其机制研究

目的：观察异丙酚对离体大鼠心肌缺血/再灌注损伤的影响并从氧化应激和线粒体介导的凋亡方面探讨其作用机制。方法：应用Langendorff离体心脏灌注系统建立心肌缺血/再灌注损伤模型。40只SD大鼠随机分为正常对照组、缺血/再灌注模型（I/R）组、异丙酚15、30、60μmol.L-1组。除正常对照组外,各组分别平衡灌注20 min后,常温全心停灌25 min,再灌注30 min。Powerlab/8s仪记录各组平衡末、缺血前及再灌30 min时的各项心功能指标并测定冠脉流出液中乳酸脱氢酶（LDH）、肌酸激酶（CK）活性;检测心肌线粒体活力、膜肿胀度、锰超氧化物岐化酶（Mn-SOD）活性和丙二醛（MDA）含量;流式细胞仪检测心肌细胞凋亡;流式细胞术检测Bcl-2和Bax的表达,免疫组化法测定天冬氨酸特异的半胱氨酸蛋白酶（caspase）-3,9,8蛋白的表达。结果：与I/R组相比,异丙酚30、60μmol.L-1能明显改善缺血/再灌注后的心功能,减弱冠脉流出液中LDH、CK的活性（P〈0.05）;心肌线粒体活力有所恢复,膜肿胀度减轻,Mn-SOD活性升高,MDA生成明显减少（P〈0.05）,心肌细胞凋亡明显减少,Bcl-2表达上调,Bax表达下调,caspase-3,9阳性表达细胞数明显减少（P〈0.05）。结论：异丙酚明显减轻缺血/再灌注所致的心肌线粒体的过氧化损伤,抑制线粒体途径的凋亡,可能是其心肌保护作用机制之一。     

大鼠心肌缺血再灌注损伤模型的实验研究

目的 制备一种新型的心肌急性缺血再灌注损伤模型,以探讨一种更符合临床实际需求的实验方法.方法 将20只雌性SD(Sprague-Dawley)大鼠随机分成2组(对照组、实验组),采用结扎主动脉根部引起心肌缺血5min再灌注30 min建立心肌急性缺血再灌注模型;通过应用透射电镜观察心肌细胞超微结构的改变,同时检测心肌组织匀浆丙二醛(Maleic Dialdehyde,MDA)含量、超氧化物歧化酶(Superoxide Dismutase,SOD)活力.结果 透射电镜下超微结构显示实验组较对照组明显加重了心肌组织结构和线粒体的损害;实验组心肌组织MDA明显高于对照组(P<0.01),而SOD明显低于对照组(P<0.01).结论 本实验成功建立了方法简便、易于操作、取材范围广泛的心肌缺血再灌注损伤模型,为心肌缺血再灌注损伤研究提供了一种更为可行的模型.     

二氮嗪对大鼠供心心肌线粒体结构及通透性的影响

目的:探讨线粒体ATP敏感性钾离子通道(mitoKATPC)开放剂二氮嗪(DE)对离体大鼠供心长时程低温保存时线粒体超微结构及线粒体渗透性转换孔(MPTP)开放的影响。方法:利用Langendorff离体鼠心灌注法,观察供心在4℃含不同浓度DE(15、30、45μmol/L)的Celsior保存液中保存9h后,复灌期心脏作功量(RPP)变化情况。比色法测定MPTP开放情况;透射电子显微镜观察心肌细胞线粒体超微结构的变化。结果:①Celsior保存液中加入30μmol/L的DE对促进长时程低温保存后供心收缩功能的恢复、减轻心肌细胞线粒体超微结构损伤和抑制MPTP开放的作用最显著。②DE的上述作用可分别被mitoKATP特异性阻断剂5-羟基葵酸盐(5-HD)及MPTP开放剂苍术苷(Atr)所取消。结论:DE可通过抑制MPTP开放而减轻由长时程低温保存导致的大鼠供心心肌线粒体超微结构的损伤。     

Cell biology of cardiac mitochondrial phospholipids.

Phospholipids are important structural and functional components of all biological membranes and define the compartmentation of organelles. Mitochondrial phospholipids comprise a significant proportion of the entire phospholipid content of most eukaroytic cells. In the heart, a tissue rich in mitochondria, the mitochondrial phospholipids provide for diverse roles in the regulation of various mitochondrial processes including apoptosis, electron transport, and mitochondrial lipid and protein import. It is well documented that alteration in the content and fatty acid composition of phospholipids within the heart is linked to alterations in myocardial electrical activity. In addition, reduction in the specific mitochondrial phospholipid cardiolipin is an underlying biochemical cause of Barth Syndrome, a rare and often fatal X-linked genetic disease that is associated with cardiomyopathy. Thus, maintenance of both the content and molecular composition of phospholipids synthesized within the mitochondria is essential for normal cardiac function. This review will focus on the function and regulation of the biosynthesis and resynthesis of mitochondrial phospholipids in the mammalian heart.     

Lipid peroxidation and alterations to oxidative metabolism in mitochondria isolated from rat heart subjected to ischemia and reperfusion.

Ischemia-reperfusion injury to cardiac myocytes involves membrane damage mediated by oxygen free radicals. Lipid peroxidation is considered a major mechanism of oxygen free radical toxicity in reperfused heart. Mitochondrial respiration is an important source of these reactive oxygen species and hence a potential contributor to reperfusion injury. We have examined the effects of ischemia (30 min) and ischemia followed by reperfusion (15 min) of rat hearts, on the kinetic parameters of cytochrome c oxidase, on the respiratory activities and on the phospholipid composition in isolated mitochondria. Mitochondrial content of malonyldialdheyde (MDA), an index of lipid peroxidation, was also measured. Reperfusion was accompanied by a significant increase in MDA production. Mitochondrial preparations from control, ischemic and reperfused rat heart had equivalent Km values for cytochrome c, although the maximal activity of the oxidase was 25 and 51% less in ischemic and reperfused mitochondria than that of controls. These changes in the cytochrome c oxidase activity were associated to parallel changes in state 3 mitochondrial respiration. The cytochrome aa3 content was practically the same in these three types of mitochondria. Alterations were found in the mitochondrial content of the major phospholipid classes, the most pronounced change occurring in the cardiolipin, the level that decreased by 28 and by 50% as function of ischemia and reperfusion, respectively. The lower cytochrome c oxidase activity in mitochondria from reperfused rat hearts could be almost completely restored to the level of control hearts by exogenously added cardiolipin, but not by other phospholipids nor by peroxidized cardiolipin. It is proposed that the reperfusion-induced decline in the mitochondrial cytochrome c oxidase activity can be ascribed, at least in part, to a loss of cardiolipin content, due to peroxidative attack of its unsaturated fatty acids by oxygen free radicals. These findings may provide an explanation for some of the factors that lead to myocardial reperfusion injury.     

克山病病区粮低Se对大白鼠心肌线粒体Ca转运影响的研究

以低Ｓｅ克山病病区粮喂养大白鼠为动物模型,在细胞及亚细胞水平上进行了低Ｓｅ与Ｃａ转运关系的研究,同时测定了线粒体的能量转换功能。结果显示,低Ｓｅ病区粮组动物心肌线粒体Ｃａ转运呈现明显异常,但线粒体能量转换功能尚未发生明显改变。提示线粒体Ｃａ转运功能损伤先于线粒体能量转换功能损伤之前发生。心肌线粒体Ｃａ转运功能可作为更灵敏的指标用于克山病发病机理的研究。上述结果进一步表明克山病是一种“心肌线粒体病”。     

Adenine nucleotide translocase activity and sensitivity to inhibitors in hepatomas. Comparison of the ADP/ATP carrier in mitochondria and in a purified reconstituted liposome system

Adenine nucleotide uptake was found to be lower in mitochondria from hepatoma 7777, 7800, and 9618A than in the host livers. Moreover, in the fast-growing hepatoma 7777 the sensitivity of the adenine nucleotide translocase to inhibition by carboxyatractylate and bongkrekic acid was considerably decreased. Purification of the ADP/ATP carrier from hepatoma 7777 mitochondria and its reconstitution into an artificial liposome system reversed the abnormal kinetics in that the adenine nucleotide uptake and response to inhibitors were identical in proteoliposome preparations from host liver and tumor mitochondria. Analysis of the lipids of the hepatoma inner mitochondrial membrane indicated considerable differences from normal in the levels of phospholipids and cholesterol. Most striking was the increase in cholesterol and sphingomyelin of the hepatoma 7777 inner membrane. An artificial liposome system containing cholesterol in addition to the standard phospholipids could produce alterations in kinetics of the purified ADP/ATP carrier from heart mitochondria similar to those seen in the hepatoma 7777. In general, these results support the suggestion that alterations in the lipid environment of the inner mitochondrial membrane rather than intrinsic changes in the carrier protein itself produce the aberrant observations of adenine nucleotide translocase activity in hepatoma mitochondria.     

Phospholipid composition of highly purified mitochondrial outer membranes of rat liver and Neurospora crassa. Is cardiolipin present in the mitochondrial outer membrane?

Isolated mitochondrial outer membrane vesicles (OMV) are a suitable system for studying various functions of the mitochondrial outer membrane. For studies on mitochondrial lipid import as well as for studies on the role of lipids in processes occurring in the outer membrane, knowledge of the phospholipid composition of the outer membrane is indispensable. Recently, a mild subfractionation procedure was described for the isolation of highly purified OMV from mitochondria of Neurospora crassa (Mayer, A., Lill, R. and Neupert, W. (1993) J. Cell Biol. 121, 1233–1243). This procedure, which consists of swelling and mechanical disruption of mitochondria followed by two steps of sucrose density gradient centrifugation, was adapted for the isolation of OMV from rat liver mitochondria. Using the appropriate enzyme markers it is shown that the resulting OMV are obtained in a yield of 25%, and that their purity is superior to that of previous OMV preparations. Analysis of the phospholipid composition of the OMV showed that phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol are the major phospholipid constituents, and that cardiolipin is only present in trace amounts. The phospholipid composition is very similar to that of the highly purified OMV from mitochondria of Neurospora crassa, although the latter still contain a small amount of cardiolipin.     

Role of monoaminoxidase in the intensification of peroxidation of lipids in mitochondria during experimental myocardial necrosis

The relationship between lipid peroxidation and rat heart mitochondrial monoamine oxidase activity was studied in experimental myocardial necrosis induced by adrenaline injection. It has been established that both the intensity of peroxidation and the activity of monoamine oxidase in mitochondria from adrenaline-injured rat myocardium were essentially increased. The preliminary administration of antioxidants (vitamin E and ionol) was shown to decrease both the intensity of lipid peroxidation and the activity of monoamine oxidase. It is suggested that intensification of lipid peroxidation which is considered to be the main pathogenic factor in ischemic myocardial injury depends on mitochondrial monoamine oxidase activity. Protective effects of antioxidants are realized by the action on two subsequent chains during the formation of active oxygen forms and destruction of lipid peroxidation products.     

Stilbene derivatives inhibit the activity of the inner mitochondrial membrane chloride channels

Ion channels selective for chloride ions are present in all biological membranes, where they regulate the cell volume or membrane potential. Various chloride channels from mitochondrial membranes have been described in recent years. The aim of our study was to characterize the effect of stilbene derivatives on single-chloride channel activity in the inner mitochondrial membrane. The measurements were performed after the reconstitution into a planar lipid bilayer of the inner mitochondrial membranes from rat skeletal muscle (SMM), rat brain (BM) and heart (HM) mitochondria. After incorporation in a symmetric 450/450 mM KCl solution (cis/trans), the chloride channels were recorded with a mean conductance of 155 ± 5 pS (rat skeletal muscle) and 120 ± 16 pS (rat brain). The conductances of the chloride channels from the rat heart mitochondria in 250/50 mM KCl (cis/trans) gradient solutions were within the 70–130 pS range. The chloride channels were inhibited by these two stilbene derivatives: 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) and 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid (SITS). The skeletal muscle mitochondrial chloride channel was blocked after the addition of 1 mM DIDS or SITS, whereas the brain mitochondrial channel was blocked by 300 μM DIDS or SITS. The chloride channel from the rat heart mitochondria was inhibited by 50–100 μM DIDS. The inhibitory effect of DIDS was irreversible. Our results confirm the presence of chloride channels sensitive to stilbene derivatives in the inner mitochondrial membrane from rat skeletal muscle, brain and heart cells.     

EFFECTS OF ACTINOMYCIN D AND PUROMYCIN ON THE ACTH-INDUCED ULTRASTRUCTURAL TRANSFORMATION OF MITOCHONDRIA OF CORTICAL CELLS OF RAT ADRENALS IN TISSUE CULTURE

The ultrastructure of the mitochondria of the cultured cortical cells of rat adrenals was studied. In vivo it was found that the zona fasciculata mitochondria have vesicular internal structure. 600-A vesicles appear free in the matrix or as protrusions of the inner mitochondrial membrane. In tissue cultures of the fetal and newborn rat adrenal cortex it was seen that ACTH induces transformation of the tubulo-vesicular internal structure of the mitochondria to 600-A vesicles. Actinomycin D and puromycin inhibited this transformation if they were added with ACTH. When added alone, these inhibitors of protein synthesis induced no change in the ultrastructure of the mitochondria in cultured cortical cells of rat adrenals.