组胺作为一种交感神经递质的新证据

发布时间：2018-07-22 11:10

【摘要】： 背景与目的组胺(Histamine, HA)是人体内重要的自体活性物质,具有广泛的生理作用,同时也参与介导多种疾病的病理生理过程。在中枢神经系统,存在完整的HA能神经体系,HA作为中枢神经递质参与介导认知记忆、睡眠觉醒、饮水摄食等功能。在外周组织中,HA主要以肥大细胞源和非肥大细胞源两种形式贮存。肥大细胞来源的HA参与介导变态反应的病理过程,非肥大细胞源性HA的生理和病理作用尚不十分清楚。业已发现,非肥大细胞来源的HA存在于大鼠外周胃肠道的神经、颈上神经节(Superior cervical ganglion, SCG)、颈动脉体感觉神经元等,提示HA可能与这些神经元的活动有关。我们以往的研究发现,在豚鼠SCG的神经元内,参与合成HA的组胺酸脱羧酶(Histidine decarboxylase, HDC)与合成去甲肾上腺素(Norepinephrine, NE)的酪氨酸羟化酶(Tyrosine hydroxylase, TH)和多巴胺β羟化酶(Dobamine-β-hydroxylase, DβH)共存,而且HA和NE也共存其中;刺激交感神经末梢能使HA和NE从交感神经末梢共释放;激动交感神经末梢突触前膜HA H3受体能够负反馈调控HA和NE的释放。根据以上研究结果,我们首先提出HA很可能是一种尚未被认识的新的交感神经递质,并提出HA参与负反馈调控交感神经传递的假说。然而HA与NE在交感神经元内的共存是否具有普遍性?HA在交感神经元内的亚细胞定位如何?HA从交感神经元内释放是否遵循经典神经递质囊泡释放的规律?这些都是为最终定义HA作为一种新的交感神经递质所必须回答的问题。基于以上问题,本研究采用免疫荧光多重标记、顺行示踪及激光共聚焦显微镜技术,观察HA在不同种属动物交感神经节与交感神经末梢的分布,及其与NE的共存;采用包埋前免疫电镜技术观察HA在交感神经末梢和培养交感神经节细胞的亚细胞定位;在新生豚鼠原代培养SCG细胞上,利用FM1-43荧光染料研究HA突触囊泡释放、运动的循环动力学,为最终证实HA是一种新的交感神经递质提供新证据。开展这一领域的研究对于揭示交感神经的新功能,深入认识交感神经功能紊乱疾病相关疾病的发病机理,寻找新的具有潜在治疗可能性的药物新靶点等都具有重要的理论意义。结果 1.组胺和NE在不同动物交感神经系统的共存分布本研究采用免疫荧光双标和激光共聚焦显微镜技术,发现在小鼠SCG、狗SCG和腹腔交感神经节神经元内,以及小鼠和豚鼠输精管、肠系膜动脉的交感神经神经末梢中均有HA和NE共存。在小鼠和狗的SCG中,NE样免疫阳性神经元中HA的双标率分别为70%和40%,狗腹腔节中NE样免疫阳性神经元中HA的双标率为60%。在小鼠和豚鼠的输精管、肠系膜动脉标本上,NE样免疫阳性神经纤维中HA的双标率分别为80%和30%。在豚鼠SCG局部微量注射顺行示踪剂生物素化葡聚糖胺(Biotinylated dextranamine, BDA),取豚鼠心脏标本,采用免疫荧光三重标记技术观察自SCG投射到心脏的交感神经纤维和末梢中HA。结果显示,BDA顺行示踪投射到心脏的交感神经纤维中,20%的示踪神经纤维呈HA样免疫阳性反应,10%示踪神经纤维呈现HA和NE样免疫阳性反应。上述结果在细胞水平确定HA广泛分布于不同种属动物的交感神经系统内,并与NE共存,为定义HA作为新的交感神经递质提供了直接的形态学证据。 2.组胺在交感神经末梢及培养SCG神经元中的亚细胞定位在支配豚鼠输精管交感神经末梢内,以及原代培养的豚鼠SCG神经元内,利用HA包埋前免疫电镜技术,电镜观察显示,HA样免疫阳性物质定位于直径约50 nm的小囊泡内,且HA阳性囊泡约占囊泡总数的90%以上,大囊泡(直径100 nm)及个别小囊泡无HA免疫阳性物质。结果表明,交感神经元内HA主要存在于小囊泡中,本项结果在亚细胞水平为定义HA作为新的交感神经递质提供了直接的形态学证据。 3.培养SCG神经突起部位组胺突触囊泡循环动力学利用FM1-43荧光染料,结合HA免疫荧光染色,在培养豚鼠SCG神经元突起上观察HA囊泡的释放动力学和HA囊泡的运动过程,激光共聚焦显微镜成像并统计分析结果。脱色(Destaining)研究结果显示,SCG神经突起中HA的红色荧光信号强度与FM1-43绿色荧光信号强度随除极刺激作用时间的增加,而呈现同时逐渐减弱的现象,两者呈显著正相关(r = 0.989, P 0.01),表明HA的释放遵循囊泡释放动力学的普遍规律。在除极作用的不同时间点固定细胞,分别测定代表HA的红色荧光强度和代表FM1-43的绿色荧光强度,结果表明HA的红色荧光强度要强于FM1-43的绿色荧光强度。由于在本研究采用的除极条件下,FM1-43荧光信号主要反映循环囊泡的释放情况,HA与FM1-43的荧光强度出现差异的结果提示,在神经突起部位贮存HA的囊泡可能存在可释放囊泡库和储备囊泡库之分。荧光漂白恢复(Fluorescence recovery after photobleaching, FRAP)结合免疫组织化学研究结果显示,在SCG神经突起处含有HA囊泡的扩散速度为0.09μm2/s,其他囊泡为0.08μm2/s。HA囊泡的运动过程与其他囊泡无显著性差异。结论: 1. HA普遍存在于多种动物的交感神经系统,并与经典交感神经递质NE共存。 2. HA主要贮存于交感神经突触小囊泡内。 3.交感神经元内HA囊泡的释放遵循经典递质囊泡循环动力学规律,贮存HA的囊泡可能存在可释放囊泡库和储备囊泡库之分。
[Abstract]:Background and purpose
Histamine (HA) is an important autoactive substance in the human body. It has extensive physiological function and also participates in the pathophysiological process of mediating various diseases. In the central nervous system, there is a complete HA energy system, and HA as a central neurotransmitter is involved in mediating cognitive memory, sleeping awakening, drinking water intake and so on. In the peripheral group In the fabric, HA is mainly stored in two forms of mast cell source and non mast cell source. Mast cell derived HA is involved in the pathological process of mediating allergy. The physiological and pathological effects of non mast cell derived HA are not very clear. It has been found that non mast cell derived HA exists in the peripheral nerve of the stomach and intestines of the rat, and the superior cervical ganglion (Su Perior cervical ganglion, SCG), carotid body sensory neurons, etc., suggesting that HA may be associated with the activity of these neurons. Our previous study found that in the neurons of SCG of the guinea pig, the histamine acid decarboxylase (Histidine decarboxylase, HDC) and the tyrosine hydroxylase synthase of noradrenaline (Norepinephrine, NE) were involved in the SCG of the guinea pig. (Tyrosine hydroxylase, TH) coexist with the dopamine beta hydroxylase (Dobamine- beta -hydroxylase, D beta H), and both HA and NE coexist. Stimulating sympathetic nerve endings can release HA and NE from the sympathetic nerve endings. We first suggested that HA is likely to be a new sympathic neurotransmitter that has not yet been recognized, and proposes the hypothesis that HA participates in the negative feedback regulation of sympathetic neurotransmission. However, does the coexistence of HA and NE in sympathetic neurons are universal? What is the subcellular localization of HA in sympathetic neurons and whether HA is released from sympathetic neurons to follow the classics The release of neurotransmitter vesicles? These are questions that must be answered by the final definition of HA as a new sympathetic neurotransmitter. Based on the above problems, this study uses multiple immunofluorescence markers, anterograde tracing and laser confocal microscopy, to observe the sympathetic ganglion and sympathetic nerve endings of HA in different species of animals. Distribution and coexistence with NE; the subcellular localization of HA in sympathetic nerve endings and cultured sympathetic ganglion cells was observed by pre embedding immunoelectron microscopy, and on the primary cultured SCG cells of newborn guinea pigs, FM1-43 fluorescent dyes were used to study the release of HA synaptic vesicles and the kinetics of movement, which finally confirmed that HA was a new sympathetic nerve. The research in this field is of great theoretical significance to reveal the new function of sympathetic nerve, to understand the pathogenesis of the disease related to the disorder of sympathetic nervous disorder, and to find new potential therapeutic targets for potential treatment.
Result
1. coexistence of histamine and NE in the sympathetic nervous system of different animals
In this study, immunofluorescence double labeling and laser confocal microscopy were used to detect the coexistence of HA and NE in the sympathetic nerve endings of mice and guinea pigs and the sympathetic nerve terminals of the mesenteric arteries in mouse SCG, dog SCG and abdominal sympathetic ganglion neurons, and in the SCG of mice and dogs, the double standard rate of HA in the NE like immunoreactive neurons was respectively in mice and dogs. For 70% and 40%, the double standard rate of HA in NE like immun-positive neurons in the abdominal segment of the dog was 60%. in the vas deferens of mice and guinea pigs, and on the mesenteric artery specimens, the double standard rate of HA in the NE like immun-positive nerve fibers was 80% and 30%. respectively in the local microinjection of SCG in guinea pig SCG (Biotinylated dextranamine, BDA). A guinea pig heart specimen was taken to observe the HA. results in the sympathetic fibers and terminals projected from SCG to the heart by immunofluorescence three markers. The results showed that the BDA tracer projected into the sympathetic fibers of the heart, 20% of the tracer fibers were HA like immunoreactive, and 10% of the tracer fibers presented HA and NE like immunoreaction. The results indicated that HA was widely distributed in the sympathetic nervous system of different species of animals at the cellular level, and coexisted with NE, providing a direct morphological evidence for the definition of HA as a new sympathetic neurotransmitter.
2. subcellular localization of histamine in sympathetic nerve terminals and cultured SCG neurons
In the innervation of the sympathetic nerve endings of the guinea pig's vas deferens and the primary cultured guinea pig SCG neurons, the immuno electron microscopy showed that the HA like immunoreactive substance was located in the small vesicles with a diameter of about 50 nm, and that the HA positive vesicles accounted for more than 90% of the total vesicles, and the large vesicles (diameter 100 nm) and individual small vesicles were not H. A immunoreactive substances. The results showed that HA mainly existed in the vesicles of the sympathetic neurons. This result provides a direct morphological evidence for the definition of HA as a new sympathetic neurotransmitter at the subcellular level.
3. the kinetics of histamine synaptic vesicle circulation in SCG neurite outgrowth
Using FM1-43 fluorescent dye and HA immunofluorescence staining, the release kinetics of HA vesicles and the movement process of HA vesicles were observed on the SCG neurites in the cultured guinea pigs. The laser confocal microscope imaging and statistical analysis were made. The results of decolorization (Destaining) showed that the red fluorescence signal intensity of HA in the protuberance of the SCG God and the FM1-43 green in the protuberance of the SCG God. The intensity of the fluorescence signal increased with the increase of the depolarization time, and showed a gradual weakening. Both showed significant positive correlation (r = 0.989, P 0.01). It showed that the release of HA followed the general rule of the kinetics of vesicle release. The red fluorescence intensity and FM1-43 representing the HA were determined by the fixed cells at different time points of depolarization. The results of green fluorescence intensity indicate that the red fluorescence intensity of HA is stronger than the green fluorescence intensity of FM1-43. Due to the depolarization conditions used in this study, the FM1-43 fluorescence signal mainly reflects the release of circulating vesicles. The difference between the fluorescence intensity of HA and FM1-43 suggests that the vesicles stored on the HA at the nerve sites may exist. The results of Fluorescence recovery after photobleaching (FRAP) combined with immunohistochemical study showed that the diffusion rate of HA vesicles at SCG neurites was 0.09 u m2/s, and the other vesicles of 0.08 u m2/ s.HA vesicles were not significantly worse than other vesicles. Different.
Conclusion:
1. HA is ubiquitous in the sympathetic nervous system of many animals and coexists with the classical sympathetic neurotransmitter NE.
2. HA is mainly stored in the sympathetic synaptic vesicles.
The release of HA vesicles in 3. sympathetic neurons follows the classical neurotransmitter vesicle cycle dynamics. The storage of HA vesicles may be divided into release vesicles and reserve vesicles.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R363

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