缺血/再灌注损伤后心肌细胞线粒体功能及其能量代谢变化
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摘要
研究目的:1.建立离体心脏缺血/再灌注损伤模型,监测大鼠的心功能变化并测定心肌生化指标,验证离体缺血/再灌注损伤模型建立成功。2.成功建立缺血/再灌注损伤模型后,探讨缺血/再灌注损伤后心肌细胞线粒体功能及其能量代谢变化。
研究方法:1. SD雄性大鼠脱颈椎处死,迅速取出心脏后投入到4℃的K-H液中,行主动脉插管,丝线结扎固定,挂靠在Langendorff灌流装置,连接压力换能器传感器,开始灌流;RM-6280多道生理记录和分析系统记录分析心功能变化;收集灌流后液体,测定心肌生化指标。2.将实验动物分为正常对照组、单纯缺血组和缺血再灌注组(n=8)。各组灌流结束后,取大鼠左室全层心肌作为标本提取目的蛋白AMPK和Cyt– C。Western blot法测定心肌细胞AMPKα和Cyt - C的蛋白表达。
研究结果:1.离体缺血/再灌注损伤模型结果:与对照组相比,实验组的LVSP(P <0.05)、+dp/dtmax(P <0.05)与-dp/dtmax(P <0.05)降低;LVEDP(P <0.05)升高,两组相比差异显著;而两组HR差异不显著(P >0.05)。I/R组心肌生化指标CK、LDH、CK-MB与实验组比较明显升高(P <0.05)。提示大鼠离体心脏缺血再灌注模型建立成功。2.缺血再灌注组AMPKα的蛋白表达显著降低(P<0.05)。而细胞色素C的蛋白表达均显著升高(P<0.05)。
研究结论:1.成功建立离体缺血/再灌注损伤模型。2.缺血/再灌注损伤后心肌细胞能量蛋白AMPKα1的蛋白表达显著降低,而细胞色素C的蛋白表达均显著升高。
Objective: 1. to establish in vitro ischemia / reperfusion injury model in rats to monitor changes in cardiac function and biochemical indicators of myocardial;Verify the in vitro ischemia / reperfusion injury model was established successfully. 2. Successfully established ischemia / reperfusion injury model; then test the mitochondrial function and energy metabolism.
Methods: 1. SD rats were killed off the spine, removed the heart quickly into 4℃KH solution, aortic cannulation, silk ligation fixed, anchored in the Langendorff perfusion apparatus, connecting the pressure transducer sensor, start perfusion; RM-6280 multi-channel recording and analysis system recorded the physiological analysis of cardiac function; collecting fluid after perfusion and testing the myocardial biochemical indicators. 2. The experimental animals were divided into normal control group, ischemia-reperfusion group and ischemia group (n = 8). After perfusion in each group, taking the left ventricular myocardial to extract the protein of AMPK and Cyt - C. using Western blot to determine the protein of AMPKαand Cyt - C.
Results: 1.The results In vitro ischemia / reperfusion injury model: Compared with the control group, LVSP (P <0.05), + dp / dtmax (P <0.05) and -dp/dtmax (P <0.05 ) decreased in experimental group; LVEDP (P <0.05) increased in experimental group with a significant difference; and HR groups was not significant (P > 0.05). Compared with the experimental group, I / R myocardial biochemical indicators CK, LDH, CK-MB was significantly higher (P <0.05). We can say the in vitro ischemia-reperfusion model is established successfully. 2. The expression of AMPKαprotein was significantly decreased (P <0.05) in Ischemia-reperfusion group. The expression of cytochrome C protein were significantly higher (P <0.05).
Conclusions: 1. the in vitro ischemia / reperfusion injury model was successfully established. 2. The expression of AMPKαprotein was significantly decreased (P <0.05) in Ischemia-reperfusion group, but the expression of cytochrome C protein was significantly increased.
研究方法:1. SD雄性大鼠脱颈椎处死,迅速取出心脏后投入到4℃的K-H液中,行主动脉插管,丝线结扎固定,挂靠在Langendorff灌流装置,连接压力换能器传感器,开始灌流;RM-6280多道生理记录和分析系统记录分析心功能变化;收集灌流后液体,测定心肌生化指标。2.将实验动物分为正常对照组、单纯缺血组和缺血再灌注组(n=8)。各组灌流结束后,取大鼠左室全层心肌作为标本提取目的蛋白AMPK和Cyt– C。Western blot法测定心肌细胞AMPKα和Cyt - C的蛋白表达。
研究结果:1.离体缺血/再灌注损伤模型结果:与对照组相比,实验组的LVSP(P <0.05)、+dp/dtmax(P <0.05)与-dp/dtmax(P <0.05)降低;LVEDP(P <0.05)升高,两组相比差异显著;而两组HR差异不显著(P >0.05)。I/R组心肌生化指标CK、LDH、CK-MB与实验组比较明显升高(P <0.05)。提示大鼠离体心脏缺血再灌注模型建立成功。2.缺血再灌注组AMPKα的蛋白表达显著降低(P<0.05)。而细胞色素C的蛋白表达均显著升高(P<0.05)。
研究结论:1.成功建立离体缺血/再灌注损伤模型。2.缺血/再灌注损伤后心肌细胞能量蛋白AMPKα1的蛋白表达显著降低,而细胞色素C的蛋白表达均显著升高。
Objective: 1. to establish in vitro ischemia / reperfusion injury model in rats to monitor changes in cardiac function and biochemical indicators of myocardial;Verify the in vitro ischemia / reperfusion injury model was established successfully. 2. Successfully established ischemia / reperfusion injury model; then test the mitochondrial function and energy metabolism.
Methods: 1. SD rats were killed off the spine, removed the heart quickly into 4℃KH solution, aortic cannulation, silk ligation fixed, anchored in the Langendorff perfusion apparatus, connecting the pressure transducer sensor, start perfusion; RM-6280 multi-channel recording and analysis system recorded the physiological analysis of cardiac function; collecting fluid after perfusion and testing the myocardial biochemical indicators. 2. The experimental animals were divided into normal control group, ischemia-reperfusion group and ischemia group (n = 8). After perfusion in each group, taking the left ventricular myocardial to extract the protein of AMPK and Cyt - C. using Western blot to determine the protein of AMPKαand Cyt - C.
Results: 1.The results In vitro ischemia / reperfusion injury model: Compared with the control group, LVSP (P <0.05), + dp / dtmax (P <0.05) and -dp/dtmax (P <0.05 ) decreased in experimental group; LVEDP (P <0.05) increased in experimental group with a significant difference; and HR groups was not significant (P > 0.05). Compared with the experimental group, I / R myocardial biochemical indicators CK, LDH, CK-MB was significantly higher (P <0.05). We can say the in vitro ischemia-reperfusion model is established successfully. 2. The expression of AMPKαprotein was significantly decreased (P <0.05) in Ischemia-reperfusion group. The expression of cytochrome C protein were significantly higher (P <0.05).
Conclusions: 1. the in vitro ischemia / reperfusion injury model was successfully established. 2. The expression of AMPKαprotein was significantly decreased (P <0.05) in Ischemia-reperfusion group, but the expression of cytochrome C protein was significantly increased.
引文
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[3] Linnane AW,et al. [J]. Lancet,1989,25:642-645.
[4]桑琛,李明学.衰老自由基学说和运动对抗自由基损伤的作用[J].吉林体育学院学报,2007,23(1):80-81.
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[6]Libby P, Ganz P. Restenosis revisited 2 new targets, new therapies [J].N Engl J Med, 1997, 337 (6): 4182419
[7]Young IS, McEneny J. Lipoprotein oxidation and atherosclerosis [J]. Biochem Soc Trans, 2001, 29 ( Pt 2) : 358-362
[8]朱晔斌,吴双,孔麟麟,等.氧化型低密度脂蛋白的形成及其致动脉粥样硬化的机制[J].武警医学院学报, 2009, 18 (11) : 62-64
[9]孟晓萍,王永维,崔建华,等. N-乙酰半胱氨酸对兔颈动脉粥样斑块的抑制作用[J].吉林大学学报(医学版) , 2008, 34 (6) : 1 004-006
[10] MadamanchiNR, Runge MS. Mitochondrial dysfunction in atherosclerosis[J].Circ Res, 2007, 100 (4) : 460-473
[11] Witko-SarsatV, Gausson V, Descamps-Latscha B. Are advanced oxidation protein productspotential uremictoxins [J] ?Kidney Int Suppl,2003, (84) : S11-S14.
[12] Descamp s-Latscha B, W itko-SarsatV, Nguyen-Khoa T,et al. Advanced oxidation protein p roducts as risk factors for atherosclerotic cardiovascular events in nondiabetic predialysis patients [J].Am J Kidney D is, 2005,45 (1) : 39-47.
[13] Lin SJ , Shyue SK, L iu PL, et al.Adenovirus-mediated overexpression of catalase attenuates oxLDL-induced apoptosis in human aortic endothelial cells via AP-1 and C-Jun N-terminal kinase /extracellular signal-regulated kinase mitogen-activated protein kinase pathways [J].J Mol Cell Cardiol, 2004, 36 (1) : 129-139.
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[15] Shoji M, Tsutaya S, Kasai T, et a1. Implication of single nucleotide polymorphisms in association study: mitochondrial variations as another genetic markers for hypertension[J]. Rinsho Byori, 2002, 50:4972-5011.
[16] Davidson SM, Duchen MR. Effects of NO on mitochondrial function in cardiomyocytes: Pathophysiological relevance[J]. Cardiovasc Res, 2006, 71(1):10- 21.
[17] Kojic ZZ, Flogel U, Schrader J, et al. Endothelial NO formation does not control myocardial O2 consumption in mouse heart[J]. Am J Physiol Heart Circ Physiol, 2003, 285 (1): H392 - H397.
[18] Jacobson J, Duchen MR, Hothersall J, et al. Induction of mitochondrial oxidative stress in astrocytes by nitric oxide precedes disruption of energy metabolism[J]. J Neuro chem, 2005, 95 (2) : 388 - 395.
[19] Duchen MR. Roles of mitochondria in health and disease[J]. Diabetes, 2004, 53 ( Suppl) : S96 - S102.
[20] PackerMA , Murphy MP. Peroxynitrite formed by simultaneous nitric oxide and superoxide generation causes cyclosporin-A -sensitive mitochondrial calcium efflux and depolarisation[J]. Eur J Biochem, 2005, 234 (1) : 231 -239
[21] Brookes PS, Salinas EP, Darley-Usmar K, et al. Concentration- dependent effects of nitric oxide on mitochondrial permeability transition and cytochrome c release[J].J Biol Chem, 2000, 275 (27) : 20474 - 20479.
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