【作者】 潘秉兴；

【导师】 赵克森；

【作者基本信息】 第一军医大学 ， 病理生理学， 2004， 博士

【摘要】 立项背景：既往的研究已经提示，诱导型一氧化氮合酶（inducible nitric oxide synthase，iNOS）的过量表达伴随一氧化氮（nitric oxide，NO）分子的大量产生在许多疾病的发生和发展过程中发挥着重要的作用。然而，近年的研究却发现，NO在疾病发展中的毒损作用主要不是由它自身，而是通过与超氧阴离子(superoxide，O₂^.-)反应形成过氧亚硝酸阴离子（peroxynitrite，ONOO^-）而完成。同时，我们近来的实验表明，NO的形成同样参与了重症休克低血管反应性的发生过程。用NO供体（S-亚硝基-N-乙酰青霉胺，SNAP）产生大量NO，可以引起血管平滑肌细胞超极化和细胞内钙离子浓度下降并带来血管反应性下降；但如果预先使用自由基清除剂tiron清除O₂^.-，则可明显抵消NO的上述作用。这就提示，NO在休克过程中的这种作用可能是通过产生ONOO^-而介导，但目前仍然缺乏直接的实验证据表明ONOO^-本身具有降低血管反应性的能力。另一方面，我们及其他实验室的研究结果一致提示，钾通道(K_ATP和BK_Ca通道)激活引起平滑肌细胞超极化在休克后期血管低反应性发生中具有重要的作用，如果ONOO^-形成可以降低血管的反应性，那么，ONOO^-是否可能通过激活BK_Ca通道以及促进平滑肌细胞超极化而发挥相应的作用?为了解决上述问题，我们建立了ONOO^-的人工合成装置，通过显微监视系统实时检测提睾肌血管反应性的变化，并借助于膜片钳和激光共聚焦显微镜等多种生物学技术进行了一系列的实验。 主要结果： 一、ONOO^-对大鼠提睾肌小动脉血管反应性的影响及可能的机制 1)用人工合成的ONOO^-（0～100μM）灌流处理游离的大鼠提睾肌40min，Peroxynitrite and vaseular hyPoreactivity 发现提辜肌小动脉的血管反应性（以引起小动脉收缩的去甲肾上腺素 闭浓度的负对数值，即一109困E]表示，反应性与数值成正相关）出现 了一定程度的降低，这种降低呈现出明显的时间和剂量依赖效应。以 正常Kxeb’s液作为对照持续灌流提翠肌4Omin，分别选取灌流后第0、 10、20和4Omin作为观察点，发现在整个灌流过程中，小动脉的反 应性无明显变化，分别为8.00士0.53、8.43士0.43、8.29士0.47、8.2妊0.47。 当在灌流液里加入20林M0NoO’后，小动脉反应性即可出现一定程度 的降低，在上述四个观察点分别为7.56士0.58、6.71士0.89、6.57土1.02、 5.56士0.93，但与灌流前（即灌流第Omin）比较并无显著性差异。当 灌流的ON00一浓度增加到50”M时，小动脉的反应性即可出现显著 下降，并呈现出明显的时间和剂量依赖效应，在上述四个观察点分别 为8 .33士0.29、 5.11士0.79（P<0.05）、3.8妊0.96（P<0.01）、3.56士0.86（P<0.01， 以上均与灌流前比较），用Kreb’s液洗脱灌流30min后，小动脉反应 性可以恢复到6.67士0.88。当ONOO一的灌流浓度进一步上升到100林M 时，这种效应则更为明显，在上述四个观察点，小动脉的反应性分别 为8 .30士0.21、3.25士0.62（P<0.01）、2.62士0.63（P<0.01）、 2.00士0.56（P<0.01， 以上均与灌流前比较）以恢复到5.50士1.10。相反，50林M ON0o-分解 体对小动脉的反应性无明显影响，在上述四个观察点分别为 8.86士0.21、8.33士0.42、8.16士0.54、7.83士0.60。另一方面，ONOO一持 续灌流对小动脉的口径并无明显影响。在Kreb’s液对照灌流组，灌流 前后小动脉的口径分别为42.5士5.4和43.5士5.4林m;在20林MON0o’ 灌流组，灌流前后小动脉口径分别为35.3士4.7和36.3士4.4林m:而在 50及100林M灌流组，灌流前后小动脉口径分别为44.6士7.3·和 44.0士7.2林m以及39.7士8.8和35.7士10，l卜m（各组灌流giJ’后比较p>0.1）。2)用20林M0NOO’灌流游离大鼠提翠肌40min后发现，小动脉平滑肌细 胞（arteriola:smooth musele eells，ASMCs）出现轻度的超极化，在灌 一4。Peroxynitrite and vaseular hyPoreaetivity 流第0、10、20以及40min，ASMCs的膜电位分别为一55.4士1.1、 一54.6士1 .2、一55.0士0.5以及一56.2士l.3mv，但与灌流前（即灌流第omin） 比较无显著性差异。然而，当ONOo’浓度被升高到50和100林M后， 则可发现ASMCs出现明显超极化:在上述时间点ASMCs膜电位分 别为一52.6土1.0、一55.0士1.2、一56.5士1.3（P<0.05）、一59.4士0.6mV（P<0.01） 以及一53.6士1.2、一55.1士1.8、一59.3士1.1（p<0.05）、一61.8士1.3mV（p<0，01）。 用Kreb，s液洗脱灌流20min后，ASMCs的超极化有一定恢复。在50卜:M 灌流组，ASMCs的膜电位在洗脱后恢复到一56.3士1 .lmV，而在10叩M 灌流组则可恢复到一56.4士1 .3mV。相反，正常的Kreb，s液或者 50卜LMON0o-分解体对ASMCs膜电位无明显影响。3)ONOO一引起的血管低反应性可以被KCI和TEA所恢复。与灌流前相 比，以50林M ONOO一单纯灌流40min可以引起血管反应性降低 54.4士6.7%（与灌流前比较p<0.01），而oNOO’与KCI（50林M ONOO一+30 mMKCI，40min）共同灌流仅可引起小动脉反应性降低 12.8士7.1%，与灌流前比较无显著性差异（P>0.1）;ONOO一更多还原

【Abstract】 A wide variety of studies have indicated that overexpression of inducible nitric oxide synthase (iNOS) with subsequent overproduction of NO plays a pivotal role in the pathogenesis and development of many diseases. However, recent findings have strongly implied that the cytotoxic effects, which were previously attributed to NO, are mainly mediated by ONOO", a product via the rapid reaction between NO and superoxide anion (O2 ~) rather than NO per se. Concurrently, the experimental results from our lab demonstrated that NO contributed vitally to vascular hyporeactivity during severe hemorrhagic and septic shock. A large amout of NO, which was released by a NO donor (SNAP), resulted in the ASMCs hyperpolarization and decreased intracellular calcium with low vasoreactivity. However, the NO action mentioned above could be abolished by pretreatment of tiron, a scavenger of O2 , which indicated that the participant of NO in the pathogenesis of vascular hyporeactivity might be due to ONOO" generation . On the other hand, our and other’s results have unanimously revealed that membrane potassium channels activation with subsequent membrane hyperpolarization is crucially involved in vascular hyporeactivity during the late stage of severe shock. If ONOO" itself can decrease vascular reactivity to some extent, one should then consider whether BKca channel activation with membrane hyperpolarziation represents a possible access for ONOO" to exert its deleterious actions. To these ends, we performed a series of experimentswith the aids of some outstanding biological techniques including patch-9-clamp, confocal microscopy and micro-monitoring system. The results are listed below:1. The underlying mechanisms for ONOO" modulation on rat cremaster arteriolar reactivity1) Superfusion of isolated cremaster muscle with authentic ONOO" (0~100M) for 40 minutes induced a conspicuous decrease of arteriolar reactivity (AR) in a dose- and time-dependent manner. The arteriolar reactivity, which was expressed by the negative logarithm value of norepinephrine concentration in producing constriction of creamster muscle arterioles, was monitored at 0, 10th, 20th and 40th minute respectively. Continuous superfusion with Kreb’s solution had little effect on AR and the AR values at the above time points were 8.00?.53, 8.43?.43, 8.29?.47 and 8.29?.47 respectively. 20M ONOO could only slightly but not significantly decrease AR and the AR values were 7.56 0.58 6.71 0.89 6.57 1.02 5.56?.93 at the above time points. However, when ONOO" concentration in the perfusion solution was increased to 50M, it could be clearly seen that AR was significantly decreased in a time-dependent fashion with the AR values of 8.33?.29, 5.11?.79 (p<0.05 vs that of 0 min), 3.89?.96 (p<0.01 vs that of 0 min) and 3.56?.86 (p<0.01 vs that of 0 min) at the above time points Washout of ONOO" could partially restore the decreased AR to 6.67?.88. Further elevation of ONOO" to 100M exerted more deleterious effect on AR and the AR values were decreased progressively from 8.30?.21 to 3.25?.62 (p<0.01 vs that of 0 min) at 10th min, 2.62?.63 (p<0.01 vs that of 0 min) at 20th min and 2.00?.56 (p<0.01 vs that of 0 min) at 40th min, whichcould receive partial recovery to 5.50?.10 after ONOO" washout. Bycontrast, treatment with 50 decomposed ONOO" failed to produce any influences on AR. We also found that arteriolar diameter (AD) was not affected by ONOO" administration at any of the above concentrations. The AD values were 42.8 5.4 and 43.5?.4m before and after control supefusion with Kreb’s solution, which were 35.3?.7 and 36.3?.4m respectively in 20M ONOO" perfusion group. In addition, even if ONOO" concentration in the prefusate was increased to 50 or 100M, the AR underwent little changes. Prior to 50 or 100M ONOO" treatment, AR values were 44.6?.3 and 39.7?.8um respectively, which were 44.0?.2 and 38.7?0.1 urn posterior to ONOO" application.2) Superfusion with 20M ONOO" for 40 min caused a slight and not significant hyperpolarization in ASMCs.更多还原