【作者】 刘颖；

【导师】 王永利；

【作者基本信息】 河北医科大学 ， 药理学， 2008， 硕士

【摘要】 脑血管意外（CVA）或称脑卒中（Stroke）,已经成为威胁人类生命的第三大死因。约20%的病人会在一个月内死亡,50%的幸存者也会留下严重的后遗症,只有30%的病人恢复后可以独立生活,但仍遗留有神经方面的损伤。由于出血性脑卒中死亡率很高,所以CVA病人90%是缺血性脑卒中。缺血性脑卒中主要是由于供氧不足引起的。大脑本身不能储藏营养物质,其主要营养来源有赖于脑部的血液循环。缺氧可以引起神经元损伤、能量代谢障碍、和一些神经症状,可破坏血管的功能,且容易引起严重的脑出血。作为维持细胞基本生理功能的关键酶,Na⁺, K⁺-ATP酶在缺氧中所发挥的作用越来越受到重视。当缺氧发生时,ATP的合成发生障碍,从而抑制了Na⁺, K⁺-ATP酶离子泵的功能,使细胞内钠离子浓度升高,从而导致脑水肿;高浓度的钠离子又可进一步逆转钠钙交换,从而导致细胞内钙离子浓度升高,这是缺氧是细胞出现钙超载的主要原因。Na⁺, K⁺-ATP酶是否参与了豚鼠基底动脉对于缺氧的肌原性反应的调节,此时所产生的肌原性反应是否能够对抗缺氧性舒张?这是本试验首先所要阐明的问题。其次,我们想证实低浓度哇巴因是否可以作用于Na⁺, K⁺-ATP酶,从而增强血管的肌原性反应。目的:采用微血管直径测定仪,观察哇巴因及缺氧对离体豚鼠基底动脉直径影响,从而研究豚鼠基底动脉Na⁺, K⁺-ATP酶的特性,以及低浓度哇巴因是否通过Na⁺, K⁺-ATP酶对缺氧基底动脉的直径产生调节作用。方法:急性分离250g左右雄性豚鼠基底动脉,选择无分支段0.5mm连同其前后各2mm血管,穿套于Pressure Myogragh System Model 120CP浴槽中两根极细玻璃电极上,并结扎固定,管腔给予70mmHg压力,并在实验过程中持续向浴槽内通入95%O₂⁺5%CO₂混合气。稳定一小时后分别采用表面灌流给药,观察不同浓度哇巴因对豚鼠基底动脉直径的影响,以探讨缺氧对基底动脉Na⁺, K⁺-ATP酶α亚基分布及其对哇巴因亲和力的影响, 1nM哇巴因对豚鼠基底动脉缺氧性舒张的影响、1nM哇巴因对豚鼠基底动脉肌原性反应的影响以及缺氧及低浓度哇巴因和缺氧对血管收缩剂K⁺-PSS作用的影响。结果:1血管稳定性实验本实验结果表明,完整内皮和去内皮的血管标本制备完成后,持续以常氧PSS灌流60min,灌流前后其血管直径变化分别为0.3±1.2μm和0.3±1.4μm,无显著差异（P>0.05）,且当间隔1小时前后分别给予40mM K⁺-PSS时,内皮完整组血管收缩幅度分别为51.1±5.8%和49.6±7.4%,而去内皮组则为52.3±6.2%和49.8±6.5%（P>0.05）,二者之间无显著性差别（P>0.05）,提示本实验条件下,豚鼠基底动脉反应活性至少可以保持60min。2缺氧对血管直径的影响实验结果表明,内皮完整对照组血管经常氧PSS灌流60min后,直径增加0.3±1.2μm,而内皮完整缺氧组血管经无氧PSS灌流60min后,直径增加4.8±1.7μm,增加幅度较对照组显著为大（P<0.01）。去内皮对照组血管经常氧PSS灌流60min后,直径增加0.3±1.4μm,而去内皮缺氧组血管经无氧PSS灌流60min后,直径增加5.2±1.9μm,增加幅度明显大于去内皮对照组（P<0.01）,但与内皮完整缺氧组对比无显著性差异（P>0.05）。提示在70mmHg压力下,缺氧可引起豚鼠基底动脉明显舒张,且此舒张无内皮依赖性。3低浓度哇巴因对基底动脉缺氧性舒张的影响从实验结果可以看出,常氧对照组血管灌流60min PSS后,血管直径变化-0.2±1.2μm。单纯缺氧组血管预缺氧30min后继续灌流无氧PSS 30min,血管直径增加2.5±1.0μm,显著强于常氧对照组（P<0.01）。哇巴因组血管预缺氧30min后,再灌流含10-9M哇巴因的无氧PSS 30min,血管直径增加3.0±1.1μm,也显著强于常氧对照组（P<0.01）,但与单纯缺氧组没有明显差别（P>0.05）,提示低浓度哇巴因并不影响缺氧所致豚鼠基底动脉的舒张反应。4低浓度哇巴因对缺氧基底动脉肌原性反应的影响实验结果表明,内皮完整常氧组血管以含1nM哇巴因的PSS灌流30min时,由灌流0min时的333.5±23.43μm收缩至325.17±22.98μm ,灌流60min时,收缩至323.33±22.93μm,均与灌流0min时的直径有显著差异（P<0.05）。当内皮完整缺氧组血管预先用1nM哇巴因灌流30min后,血管直径则自灌流0min时的338.9±11.4μm收缩至329.6±13.3μm,有显著性差异（P<0.05）,然后换用含1nM哇巴因的PSS继续灌流使其缺氧30min后,血管进而收缩至302.5±14.9μm,较灌流0min和30min时均有显著性差异（P<0.05或0.01）,但去内皮缺氧组的血管预先用1nM哇巴因孵育30min后,其直径只自灌流0min时的337.0±19.2μm收缩至336.1±18.0μm ,无显著性差异（P>0.05）,继续灌流含1nM哇巴因的无氧PSS 30min后,也仅收缩至328.8±23.4μm与灌流0min和30min时均无显著性差异（P>0.05）。这些结果提示,1nM哇巴因单独作用不仅可引起豚鼠基底动脉收缩,也可明显增强血管在缺氧状态下的肌原性反应,但是,后者只能发生在内皮完整的血管,表现出明显的内皮依赖性。5低浓度哇巴因对高钾所致缺氧基底动脉收缩作用的影响对照组、缺氧组及哇巴因缺氧组血管经高钾液灌流时,均在4分钟内随灌流时间延长而收缩增强,灌流高钾液后第1、2、3、4分钟时的血管收缩幅度,在对照组分别为30±17.8μm、54.4±21.9μm、68±23.7μm和72.8±25.1μm,在缺氧组分别为为40.6±16.9μm、74.8±22.7μm、86.8±21.9μm和83.6±23.4μm,且无氧灌流时血管直径的变化曲线明显左移。而哇巴因缺氧组分别为61.4±20.8μm、97.2±20.4μm、102.6±22.8μm和103.6±23.1μm,且无氧灌流时血管直径的变化曲线进一步左移。提示,单纯缺氧虽然会引起血管舒张,但有收缩剂K⁺-PSS存在时,缺氧可以增加血管对收缩剂K⁺-PSS的敏感性,低浓度哇巴因可以进一步增加缺氧血管对此收缩剂的敏感性。6缺氧对豚鼠基底动脉Na⁺, K⁺-ATP酶α亚基亲和力的影响实验结果表明,不管是在常氧状态下还是在缺氧状态下,血管收缩幅度均随哇巴因浓度升高而增大.对?OD-OUA量效曲线以两个α亚基两个结合位点模式进行曲线拟合,可得到两条最佳拟合曲线,其结果提示,无论在常氧状态下还是在缺氧状态下,豚鼠基底动脉的Na⁺, K⁺-ATP酶既存在高亲和力α亚基（α₂和/或α₃亚基）,也存在低亲和力α亚基（α1亚基）。所不同的是,在常氧情况下,高亲和力α亚基与哇巴因结合的kD值为5.3×10^-10M,低亲和力α亚基的kD值为1.26×10^-6 M;而在缺氧情况下,高亲和力α亚基的kD值为1.57×10^-7M,低亲和力α亚基的kD值为9.89×10^-6 M。由以上结果可知,缺氧可明显降低豚鼠基底动脉Na⁺, K⁺-ATP酶α亚基结合哇巴因的亲和力,且对高亲和力α亚基的影响较低亲和力α亚基更为明显,其中缺氧可使豚鼠基底动脉Na⁺, K⁺-ATP酶高亲和力α亚基对哇巴因的亲和力降低约500倍;使低亲和力α亚基的亲和力仅降低8倍。结论:缺氧可引起豚鼠基底动脉明显舒张,且此舒张无内皮依赖性,但是缺氧却可以增加血管对于收缩剂K⁺-PSS的反应性。1nM哇巴因不影响豚鼠基底动脉的缺氧性舒张,但可以增强血管的肌原性反应以及缺氧血管对收缩剂的敏感性。豚鼠基底动脉既存在Na⁺, K⁺-ATP酶高亲和力催化亚基（α₂和\或α₃亚基）,也存在低亲和力催化亚基α1亚基。缺氧可明显降低豚鼠基底动脉Na⁺, K⁺-ATP酶α亚基结合哇巴因的亲和力,且对高亲和力α亚基的影响较低亲和力α亚基更为明显。更多还原

【Abstract】 Cerebrovascular accident （CVA）, or stroke, which results in a prolonged neurological disability in adults, leads to a heavy public-health and economic burden to our society every year, and it is the third most common cause of death in most countries in the world. Approximately 20% of CVA patients die within one month; about 50% of survivors need care and supervision because of their permanent and considerable disabilities; and the remaining 30% are capable of leading an independent life, despite neurological deficits. Almost 90% of the patients are victims of ischemic CVA, and the rests are of hemorrhagic CVA, maybe because the hemorrhagic is with high lethality.Ischemic CVA is always caused by the lack of O2. The interrupted blood flow can’t deliver essential oxygen and glucose to the brain who doesn’t store nutrient substances is the received pathogenic mechanism of CVA. Stroke will lead to neurological ischemia, energetic failure and neurological symptoms, followed by tissue damage within minutes if ischemia continues. The reperfusion after ischemia advances this tissue damage and induces hemorrhagic CVA.As a key enzyme in maintaining basic physiologic function of cells, more and more researches are focused on the effect of Na⁺, K⁺-ATPase on the progress of hypoxia. When theαisoform of Na⁺, K⁺-ATPase is inhibited, the Na⁺ will be retained inside the membrane, followed by an exchange of two Ca²⁺ from extra mediator and three Na⁺ from intercellular via Na⁺/Ca²⁺ exchanger type 1 （NCX1）. This progress elevates [Ca²⁺]i and initiates an increase of myogenic tone.Whether Na⁺, K⁺-ATPase participates in regulating the generation of myogenic tone on cerebral basilar arteries （CBAs）, and whether this myogenic tone overrides hypoxic dilation, are what we want to tell firstly. And secondly, whether the activity of Na⁺, K⁺-ATPase could be influenced by 1 nM ouabain and whether this could help the CBAs generating myogenic tone.Objective: Our study is to identify the effects of ouabain in different concentration on Na⁺, K⁺-ATPase form Guinea Pigs’ Basilar Artery with Pressure Myogragh System Model 120CP, to identify the effects of hypoxia on basilar arteries, and to identify the role of Na⁺, K⁺-ATPase in the regulation of myogenic tone under hypoxia.Methods: After the isolation of the basilar arteries of Guinea pigs acutely, 2.5 mm arteries without branches were cannulated on to two very tiny glass probes, and secured gently with two fine nylon sutures. Luminal pressure was hold at 70mmHg. After the equilibration period, each kind of solution will be administered by superfusion. And then, the effects of ouabain on guinea pigs’ basilar arteries in diameter, character of Na⁺, K⁺-ATPase from basilar arteries, effects of hypoxia on basilar arteries, effects of 1 nM ouabain on hypoxic dilation and myogenic tone and effects of 1 nM ouabain on arteries when vasoconstrictor is present were tested.Results:1 Test of stability of guinea pig basilar arteryAccording to the results, the passive diameter of intact and endothelium-denuded arteries, which superfused with PSS for 60 minutes, were changed by 0.3±1.2μm and 0.3±1.4μm （P>0.05）. And the effects of 40mM K⁺-PSS on diameters prior to and post the one hour superfusion contracted the intact arteries by 51.1±5.8% and 49.6±7.4% separately and contracted the endothelium-denuded arteries by 52.3±6.2% and 49.8±6.5% separately. And there is no significance between them （P>0.05）. This indicates that the stability of basilar arteries could be maintained at least 1 hour.2 Effects of hypoxia on diameter of guinea pig basilar arteriesAccording to the results, After the 60 minutes of superfusion with PSS or O2 free PSS, the passive diameter of arteries from intact control, endothelium-denuded control, intact and endothelium-denuded groups are increased by 0.3±1.2μm, 0.3±1.4μm, 4.8±1.7μm and 5.2±1.9μm respectively. The increased passive diameters of intact and endothelium-denuded arteries are much higher than the match control arteries （P<0.05）, but there is no significant difference between them （P>0.05）. This indicates that, hypoxia could induce significant dilation on basilar arteries under 70mmHg luminal pressure and this is not endothelium dependent.3 Effects of 1nM ouabain on hypoxic dilation of guinea pig basilar arteriesAccording to the results, after 0.5 hour of hypoxia, the diameters of CBAs increased by 2.5±1.0μm,which is much higher than intact normaxia group （-0.2±1.2μm, P<0.01）. Even 1 nM ouabain was added into the O2 free solution after 30 minutes of hypoxia, this could not be changed （3.0±1.1μm, P<0.01）. This indicates that, 1nM ouabain has no effects on hypoxic dilation.4 Effects of 1nM ouabain on diameter of guinea pig basilar arteriesAccording to the results, during an hour of superfusion, intact arteries could contract from 333.5±23.43μm to 323.33±22.93μm when 1nM ouabain is present. And when there is an incubation with 1nM ouabain prior to hypoxia, the intact arteries contracted from 338.9±11.4μm to 302.6±14.9μm （P<0.05）. But in the endothelium-denuded group, arteries contracted only from 337.0±19.2μm to 328.8±23.4μm （P>0.05）. This indicates that, 1nM ouabain could help CBAs generate myogenic tone not only under normaxia, but also under hypoxia, but this happened on intact basilar arteries, which presents endothelium dependence.5 Effects of 1 nM ouabain on basilar artery contraction induced by O⁺2-free K⁺-PSSWhen a high concentration of K⁺ was present, the contraction went further along with the superfusion in about 4 minutes. At the first, second, third and forth minute of superfusion, basilar arteries in control group contracted by 30±17.8μm, 54.4±21.9μm, 68±23.7μm and 72.8±25.1μm, separately. And the basilar arteries in hypoxia group contracted by 40.6±16.9μm, 74.8±22.7μm, 86.8±21.9μm and 83.6±23.4μm. This is a faster and higher contraction than control group. In ouabain-hypoxia group, basilar arteries contracted by 61.4±20.8μm, 97.2±20.4μm, 102.6±22.8μm and 103.6±23.1μm. This is a much faster and higher contraction both than control group and hypoxia group. This indicates that, hypoxia could help hypoxic arteries increase the sensitivities to vasoconstrictor K⁺-PSS, and 1 nM ouabain could further this effect.6 Effect of hypoxia on the affinities of Na⁺, K⁺-ATP aseαisoforms in guinea pig basilar arteryAccording the effects of ouabain in different concentration on passive diameter of basilar arteries under normaxia, the amplitude of contraction went further when the concentration of ouabain went higher. The contraction effects were calculated by two isoforms with two combine domain formula. The results of the calculation indicates that, there are two kinds ofαsubunits, which are with high affinity （α₂ and/orα₃） or low affinity （α₁）, present in Guinea pigs’basilar arteries. But the kD ofαisoforms are different between under normaxia and hypoxia: kD ofαisoforms with high affinity are 5.3×10^-10M and 1.57×10^-7M respectively, and kD ofαisoforms with low affinity are 1.26×10^-6 M and 9.89×10^-6 M respectively. This indicates that: 30min of hypoxia could inhibit the affinity of allαsubunits, and the inhibition onαsubunits with high affinity （α₂ and/orα₃） is more significantly.Conclusion: The isoforms ofαsubunit from guinea pigs’ CBAs areα₁ with low sensitivity andα₂ &/orα₃ with high sensitivity. 1 nM ouabain could not influence hypoxia dilation, but it could help the CBAs generate myogenic tone if there is a pre-incubation. hypoxia could help hypoxic arteries increase the sensitivities to vasoconstrictor K⁺-PSS, and 1 nM ouabain could further this effect. Hypoxia induces hypoxic dilation and depress the activities of allαisoforms from Na⁺, K⁺-ATPase, especially theαisoform with high affinity.更多还原