【作者】 杨华伟；

【导师】 邹移海； 李伊为；

【作者基本信息】 广州中医药大学 ， 中西医结合基础， 2002， 硕士

【摘要】 背景与目的 几十年来，我国对经络的研究一直是围绕两条主线来进行，一是循经感传现象的研究，一是经穴与脏腑相关途径的研究。从经络的临床应用来说，经穴是应用的核心，是经络学说的核心。关于经络实质的研究，并不仅仅在于得出经络是什么，更重要的是弄清楚经络系统的基本功能单位——腧穴是怎样和内部器官联系起来的，即经穴与脏腑的相关及其联系途径。其中研究最多的是足三里与胃经的相关途径。足三里是足阳明胃经的合穴，与胃在生理病理上有着密切的联系。实验证明电针大鼠“足三里”对应激性胃粘膜损伤具有保护作用，对其作用机制研究也是揭开穴位与脏腑相关途径的希望所在。 应激是机体对外界长久，剧烈的刺激引起的一系列非特异性反应。应激的基本反应为一系列的神经内分泌改变，最主要的神经内分泌改变是下丘脑——垂体——肾上腺皮质轴(HPA)和蓝斑——去甲肾上腺素神经元／交感——肾上腺髓质轴的强烈兴奋。其中下丘脑和蓝斑是这两个系统的中枢位点。孤束核是内脏感觉核，与内脏的运动神经核迷走神经背核关系密切，共同组成“迷走感觉运动中枢”调节胃肠运动。 内源性一氧化氮(NO)是一种新型的神经递质，在中枢神经系统一些核团内广泛存在，与一些经典的神经递质共存。内源性NO是由一氧化氮合酶(NOS)催化底物L-精氨酸L-arginine)而成，NOS的活性决定了NO的生成量。位于神经元内的结构型NOS即是长期以来用组织化学方法显示的还原型尼克酰胺腺嘌岭二核苷脱氢酶(NADPH-d)，因而一般认为用NADPH-d组化方法所显示的神经元即反映了NOS阳性结构的存在。有人报道应激能引起脑内NOS含量的升高，而应激前电针足三里可以抑制这种升高，说明电针的作用过程中有NO的参与。本实验的目的是探讨在应激时下丘脑室旁核、孤束核和蓝斑核内NOS的表达，以及电针是三里对其的调节作用，从而揭示中枢NO能神经元是足三里与胃相关联系途径中的一个重要作用。 方法 SD大鼠60只，随机分为A组：空白对照组；B组：应激性胃溃疡组；C组：电针“足三里”组；D组：L-NAME组；E组：电针“足三里”加L-NAME组；共五组，每组12只动物。模型采用束缚—水浸应激模型，用改良的GU山法计算胃粘膜溃疡指数山D。脑部切片用＊AD＊H一d法染色，光学显微镜下用北京航空航天大学病理图像分析系统分析室旁核、孤束核和蓝斑核NOS染色的积分光密度。用SPSS10．0软件包对结果进行单因素方差分析统计。 结果①大鼠在应激 7h后，胃粘膜 m为 15．42与对照组有在非常显著性差异（P＜0．01），而电针足三里后再应激，胃粘膜UI为5石7，与应激组相比，亦有非常显著差异（Pwto，of）。L—NAME组 UI为 9．58与应激组有非常显著差异（P＜0．ofX 说明 NOS抑制剂对胃粘膜亦有保护作用。L—NM组分别与电针组和电针加 L—NAME组相比，有非常显著差异（P＜0．of），说明 L—NAME虽然对胃粘膜具有保护作用，但其保护作用的效果不及电针足三里。这也许是因为电针足三里保护胃粘膜的过程中，不仅仅有NO的参与，还有其它递质参与，或者，体内不同部位的NO对胃粘膜的影响不同而引起的。②室旁核NOS表达：正常对照组有一半的细胞NOS染色阳性，染色中等程度。应激组积分光密度与对照组有非常显著差异（P＜001），电针组积分光密度与正常对照组之间无明显差异（P＞005），与应激组有非常显著差异（P＜0刀1），说明电针足三里对胃粘膜具有保护作用的同时，室旁核NOS的活性亦有变化，电针使室旁梭内增加的＊O下降。L一＊A：NO3组与对照组没有显著差异（P＞005），与应激组有非常显著差异（P＜O01）。而电针加L一＊AME组，与对照组没有显著差异o＞O刀5），与应激组比较则有极显著差异（Prto刀1）。说明电针对室旁核内NOS的表达的调节作用只在应激反应时才显现，而不能减少基础的室旁核内NOS的表达。③孤束核NOS表达：在正常对照组淡染或中度染色，应激后孤束核内细胞深染，与对照组有非常显著差异（P＜0刀1）。电针后细胞内染色变淡与应激组有极显著差异（P＜0．01），与对照组则无差异（P＞0．05）。L—NAnth组孤束核内NOS阳性神经元的染色呈淡蓝色，与对照组和电计组无明显差异（P＞0刀引，与应激组有非常显著差异（P＜0．of）。电针加 L—NAME组与 L—NAME组有非常显著差异owt001＊④蓝斑核NOS表达：正常对照组蓝斑细胞深染，应激后细胞染色稍有加深，但与正常对照组比无显著差异r＞o05），说明应激时，蓝斑核内NOS神经元无明显反应。电针组蓝斑核内积分光密度与对照组和应激组均无显著差异（P＞005）。说明电针对蓝斑核内NOS无明显影响。L—NAME组和电针加 L—NAME组与前三组相比，无明显差异o＞005）。说明蓝斑核内的 NOS 阳性神经元没有参与应激反应及电针足三里对应激的调节。 结论 电针对应激性胃粘膜损伤具有良好的保护作用，这种作用与电针对应 激时室旁核、孤束核内NO能神经元中NOS的调节有关，与蓝斑核内的NO能 神经元NOS无关。足三里与胃相关的中枢途径中有?更多还原

【Abstract】 The effect of electro-acupuncture at Zusanli Point on Nitric Oxide Synthase in the nucleis of Central Nervous System of ratsmodel of stress ulcerBackground and Objective Zusanli Point is a point of the stomach channel of foot-yangming. Acupuncture at Zusanli Point is a effective method to treat disease of gastrointestinal tract such as gastric ulcer, gastritis. There must be some connective links between Zusanli and stomach. The studies had done before have proved that electro-acupuncture at Zusanli Point can prevent the stress ulcer of rats. But how the electro-acupuncture does its effect is unknown. There are many researchers who studied on how the electro-acupuncture does its effect believed that the neurotransmitter in central nervous system(CNS) should be the important substance. The stress is a long strong response which happened while organism suffer from the harmful stimulus. The hypothalamus paraventricular nucleus(PVN) and nucleus tractus solitarii(NTS) are the CNS point which regulate the stress response. Locus ceruleus, the nucleus of sensory nerve of viscera, is also relation to stress.Endogenous nitric oxide(NO), a new style neurotransmitter, has been found in many nuclei in CNS such as PVN, NTS and LC. NO is produced by nitric oxide synthase(NOS) which catalyse the substrateL-arginine. So the content of NO depends on the activity of NOS. NOS can be displayed by histochemical method of nicotinamide adenosine dinucleotide phosphate desaturase (NADPH-d). Some researchers have proved that the content of NOS in brain of rat has remarkable rise after the rats had been stressed for 7 hours and the rise can be prevented by electro-acupuncture atZusanli before stress. The objective of this study is to observe the effect of electro-acupuncture at Zusanli on Nitric Oxide Synthase in the nucleus of CNS of rats model of stress ulcer including of PVN, NTS and LC.Methods Sixty SD rats , were divided into five groups. Group A: blank control group; Group B: stress group; Group C: electro-acupuncture at Zusanli group; Group D: L-NAME group; Group E: electro-acupuncture at Zusanli and L-NAME group, we used the restraint-immersing water model of rats and Guth’s method to calculate the Ulcer Index(UI). The brain slices were dyed by histochemical method(NADPH-d) then observed and analyzed with the image analysis system which is produced by the Beijing university of aeronautics and astronautics.Results ㏕he gastric UI of rats in stress group is more severe than blank control group, electro-acupuncture group, L-NAME group and electro-acupuncture and L-NAME group. The result shows that the electro-acupuncture at Zusanli is a efficient method to prevent stress ulcer.㏕he express of NOS in PVN: The integral optical density(IOD) of slice in stress group is remarkable different from the IOD in blank control group, electro-acupuncture group, L-NAME group and electro-acupuncture and L-NAME group. That result also shows that the increased express of NOS induced by stress can be inhibited by electro-acupuncture at Zusanli just before stress. ㏕he express of NOS in NTS: The IOD of NTS which is a sensory nerve nucleus yet is as same as that of PVN. It can be supposed that the prevention to stress ulcer of electro-acupuncture at Zusanli inhibit the increased NOS which is induced by input of impulse caused by gastric ulcer. ㏕he express of NOS in LC: The IOD of LC in each group is non-significant difference. It shows that the NO positive neuron is not relative to the stress reaction and the regulation of electro-acupuncture.Conclusion Electro-acupuncture at Zusanli Point can prevent the stress ulcer of rats. NO positive neurons in PVN and NTS have played an important role in this process. It is NO positive neuron in PVN and NTS that is the one of connective links between Zusanli and stomach.更多还原