【作者】 代政学；

【导师】 贾国良；

【作者基本信息】 第四军医大学 ， 内科学（心脏内科）， 2001， 博士

【摘要】 一氧化氮（NO）作为内皮源性舒张因子（EDRF）的主要形式，在心血管系统的生理和病理生理中发挥着重要的作用。在脓毒败血症中发生心肌收缩功能降低与心肌中NO水平增高有关。在含巨噬细胞的培养液中单个心肌细胞收缩功能降低也与NO水平增高有关。NO可产生负性肌力作用，增强舒张功能，NO是在一氧化氮合成酶（NOS）的催化下，L-精氨酸（L-Arg）和氧分子作用生成NO和L-胍氨酸。NOS分为两大类，即原生型NOS（cNOS）和诱生型NOS（iNOS）。iNOS可被一些细胞因子如白介素-1β（IL-1β）、肿瘤坏死因子（TNF-α）和γ干扰素（IFN-γ）诱导激活。细胞因子诱导产生的NO降低心肌收缩力，对心肌细胞是有毒性作用的。但是，细胞因子诱导心肌细胞死亡的确切机制尚不清楚。为了详细了解其机制，我们用培养的乳鼠心肌细胞为研究对象，观察了细胞因子（IL-1β，TNF-α、IFN-γ）对乳鼠心肌细胞有无凋亡效应。IL-1β呈时间及浓度依赖性诱导心肌细胞凋亡。本实验通过TUNEL染色，DNA琼糖脂凝胶电泳方法，流式细胞仪等方法均证实IL-1β诱导心肌细胞凋亡。细胞凋亡发生前iNOS表达增加，培养液中NO代谢产物（NOX）大量释放（5-8nmol/kg protein）。TNF-α和IFN-γ本身不能引起心肌细胞凋亡和NOX增加。IFN-γ可以使IL-1β引起心肌细胞凋亡发生提前。IL-1β引起的心肌细胞凋亡作用可被NO合成酶抑制剂所阻断。IL-1β引起的心肌细胞凋亡通过NO而起作用。 NO通过引起心肌细胞凋亡而发挥其素效应。NO供体（GSNO）呈时间及浓度依赖性引起心肌细胞凋亡。GSNO及细胞因子引起的细胞凋亡作用可以被抗氧化剂（N-acetykysteine和DTT）及半胱氨酸蛋白酶抑制剂ZVAD-fmk所中和。而cGMP依赖蛋白激动阻断剂KT5823不能阻断NO介导的细胞周亡，它们本身无细胞毒效应。亚甲蓝（MB）也可以阻断NO介 第四军医人学博士学位论文 一 导的心肌细胞凋亡，这种阻断作用是通过氧自由基的产生导致NO的减少 引起的。本实验表明NO诱导的心肌细胞凋亡需NO的氧化产物，随后激 话Caspases，最后导致心肌细胞凋亡。 IL－16在实验中证实能诱导心肌细胞凋亡。碱性成纤维细胞生长因子 （bFGF）早在 80年代末被发现，它是机体组织提取物中一种能促进生长的 活性物质，具有心肌保护功能。本实验研究bGFG是否能抑制IL．15诱导 心肌细胞凋亡及其机制，从而保护心脏功能；并测定其对P53、bgy、FSS、呼 C－myc及bclZ等几种基因产物水平的影响。本实验表明bFGF可以阻断IL1 p介导的心肌细胞凋亡作用。ILIp诱导心肌细胞凋亡的相关基因主要为 P53、Fas及C－myc。bGFF可以阻断几－1日诱导的P53、Fas及C－myc基因 表达而实现其心肌保护作用。更多还原

【Abstract】 Nitric oxide, a short-lived free radical-generating gas, is an important signal-transduction molecule in cardiovascular system cell bypes, regulalating such diverse functions as vasomotor tone, neurotransmission, mediation of immune response. In the cardiac myocyte, the major physiological rose of NO appears to involve depression of contractility and electrophysiological stabilization through elevation of intracellular cGMP. A rise in myocardial NO levels may account for the contractile depression observed during sepsis, and in isolated cardiac myocytes following exposure to macrophage-conditrioned medium. No is generated by 2 different enzymes in myocardium, which are macrophage-type, or inducible No synthase(iNOS), and endothelial-type NO synthase(eNOS). Macrophage-derived cytokines have been shown to exert negative inotropic and toxic effects on cardiac myocytes through the transcriptional activation of iNOS and subsequent production of NO iNOS is strongly induced in cardiac myocytes exposed to macrophage-derived cytokines, induding interleukin(IL)-l P tumor necrosis factor(TNF)- a and interferon(INF)- y .However, the mechanisms of cytokine-induced cardiac cardiac myocyte cell death are nuclear. To analyce these mechanisms in detail, we treated cultured neonatal cardiac myocytes in serum-free culture with a combination of the macrophage-derived cytokines interleukin-l P, tumor necrosis factor- a, and interferon- y . IL-i P caused a time-dependent and concentration-dependent -4- induction of cardiac myocyte apoptosis, beginning 72 hours after treatment, as determined by nuclean morphology, DNA internucleosomal cleavage, reflecting caspase activation. Apoptosis was preceded by a 750-fold induction of inducible NO synthase mRNA and the release of large amounts (5-8nmoll i~ g protein)of NO metabolites(NOX) into the medium. Interferon- Y and tumor necrosis factor- a had no independent effects on apoptosis or on NOX production. However, IFN- y accelerates the onset of apoptosis induced by IL-i P ,IL-l P -induced 4- myocyte apoptosis was completely blocked by an NO synthase inhibitor(L-NMMA). IL-I induced myocyte apoptosis through NO production. NO is the effector of cytokine-mediated apoptosis and exerts its effects through several mechanisms. The NO donor S-nitrosoglutathione(GSNO) caused a time-dependent and concentration-dependent induction of cardiac myoeyte apoptosis. Oxidative stress is a potent inducer of apoptosis, and redox state influences both cardiac myocyte signal transduction and gene transcription. The present studies argue in favor of an oxidative, rather than a cOMP-dependent, mechanism for cytokirie/NO-mediated apoptosis in neonatal rat cells. Most importantly, KT5823, a cGMP-dependent protein-i inhibitor, failed to prevent cytokine /NO,mediated apoptosis, while having to independent toxic effects. The inhibition of cytokine-mediated apoptosis by methylene blue can be directly attributed to quenching of NO. Since the degree to which NO forms peroxynitrite is dependent on the availability ox oxygen free radicals, the redox state of individual myocytes may be an important variable in determing the apoptotic response to NO. This possibility is supported by the fact that the antioxidants NAC and DTT were each able更多还原