锌离子在交感干的轴突运输以及锌缺乏对交感干结构影响的研究

发布时间：2018-05-13 23:23

本文选题：锌离子 + 交感干　；参考：《中国医科大学》2008年硕士论文

【摘要】： 前言锌是人体内重要的微量元素之一,是正常生长发育、基因表达、蛋白质代谢、免疫功能等过程中所必需的元素。近年来,人们对锌与神经系统生长发育以及功能调节之间的关系进行了广泛的研究,发现锌缺乏可以引起神经系统多种疾病的发生。在中枢神经系统中,大部分锌离子与蛋白质结合并维持蛋白质分子的三级结构,小部分的锌离子以游离方式存在于含锌神经元(zinc-enriched neuron,ZEN)轴突终末的突触小泡中。在突触活动中锌离子与谷氨酸、γ-氨基丁酸(gamma-aminobutyric acid,GABA)等神经递质共同释放到突触间隙,参与学习和记忆的形成、神经系统发育以及运动和感觉功能的调节。锌离子也存在于周围神经系统中,有文献报道几乎所有的颈上神经节(superior cervical ganglia,SCG)神经元中均有锌离子分布,电镜观察游离锌离子主要聚集在颈上神经节交感节后神经元核周的高尔基复合体和囊泡内,锌离子可能参与了神经元内某些含锌蛋白质的合成与组装,并维持某些蛋白质的三级结构。在脊神经节以及坐骨神经中锌离子也有广泛分布。然而,锌离子在周围神经系统神经元内的聚集转运机制以及功能尚不清楚。本研究对锌离子在交感干内的轴突运输以及锌缺乏对交感干神经纤维结构的影响进行了详细的研究。材料与方法 1、Wistar大鼠,体重约150g,雌雄不限,随机分成3组。 (1)第一组进行单侧交感干钳夹手术,对侧未做手术作为对照,分别使其术后存活2、4、6小时,在此期间注射硒化钠,然后灌流取材,采用金属自显影(AMG)的方法观察锌离子在交感干中的轴突运输情况。 (2)第二组采用与上述同样的手术方法,灌流取材后应用AMG电镜的方法观察锌离子在交感干中的分布情况。 (3)第三组大鼠钳夹单侧交感干,在靠近钳夹点的上方注射硒化钠,同样另一侧未钳夹作为对照,24h后灌流取下两侧颈上神经节进行AMG染色,观察交感干中锌离子的逆行运输。 2、生后3周的CD-1小鼠随机分为正常组和缺锌组(ZnD)组,分别给予正常饲料和缺锌饲料喂养,5周后灌流取交感干,应用常规电镜技术观察交感干的结构改变,并在光镜下比较有髓神经纤维和无髓神经纤维的数量变化,应用t检验比较两组数据之间的差异。实验结果 1、交感干钳夹后,AMG实验结果中可以看到在两个钳夹点的远端和近端有AMG阳性颗粒的蓄积,此颗粒主要位于靠近钳夹点处,但是在两个钳夹点之间没有阳性颗粒的存在,并且AMG阳性颗粒随着手术后大鼠存活时间的延长而增多。 2、AMG电镜结果显示在无髓神经纤维中有锌离子的AMG阳性反应产物。 3、在交感干逆行运输的AMG实验结果中,光镜下观察颈上神经节中仅有约5%的神经元有锌离子阳性反应产物,弥散分布于神经元的胞质内,细胞核周围染色较深,细胞核未见阳性反应,一些神经元突起内可见点线状的AMG阳性颗粒。 4、锌缺乏后导致小鼠交感干超微结构的改变,表现为髓鞘变性、排列不规则、界限不清楚,神经纤维束膜萎陷,神经轴突的横截面积减小,形态异常的神经纤维数量增多。光镜下观察,缺锌小鼠交感干有髓神经纤维的数量减少,无髓神经纤维增多。讨论有文献报道,坐骨神经钳夹后通过AMG染色方法观察到AMG阳性反应颗粒存在于钳夹点两端,说明坐骨神经钳夹点的远侧端和近侧端有锌离子的蓄积,提示在坐骨神经中存在锌离子的轴突运输,电镜下观察缺锌情况下的坐骨神经超微结构,发现有髓神经纤维的数量和表面积与正常饲养的动物相比都有所减少,髓鞘破坏变得不规则,并且发生变性,这些都证明锌可能对周围神经的相关功能具有一定的作用,因而我们推测交感干中也有锌离子的运输,并且对其结构的保持及功能的发挥起到关键性作用。应用AMG和AMG电镜技术,本实验证实了游离锌离子在交感干的轴突内进行轴突运输。研究发现,钳夹手术后,注射硒化钠,在钳夹点的两侧端均有不同程度的锌离子蓄积,并且有随着手术后存活时间的延长锌离子蓄积增多的现象,提示大多数或全部交感节后含锌神经元胞体内的锌离子可能通过顺行运输到达轴突终末并参与某些生理功能的调节,而且也有锌离子由轴突终末向胞体逆行运输。应用AMG技术检测交感干注射硒酸钠24h后,锌离子在颈上神经节中含锌神经元的表达,光镜下结果显示,颈上神经节中约有5%神经元有AMG阳性反应产物,此结果进一步证实了锌离子在交感干中进行逆行的轴突运输。结论 1、交感干存在着锌离子的顺行和逆行运输,在手术后的大鼠交感干的染色切片中可以看到均匀的AMG阳性反应。 2、缺锌后交感干结构和数量的变化说明锌离子可能对周围神经纤维结构的保持及功能的发挥起到重要的作用。
[Abstract]:Preface
Zinc is one of the important trace elements in human body. It is an essential element in normal growth, gene expression, protein metabolism and immune function. In recent years, the relationship between zinc and nervous system growth and function regulation has been extensively studied. It is found that zinc deficiency can cause a variety of diseases in the nervous system. In the central nervous system, most of the zinc ions are combined with protein and maintain the three structure of the protein molecules, and a small fraction of the zinc ions exist in the synaptic vesicles free from the end of the zinc-enriched neuron, ZEN axon. Acid, GABA) and other neurotransmitters together release into synaptic space, participate in the formation of learning and memory, the development of nervous system and the regulation of motor and sensory function. Zinc ions also exist in the peripheral nervous system. There are reports that almost all superior cervical ganglia (SCG) neurons have zinc ion distribution in almost all of the neurons of the cervical ganglion (SCG). The free zinc ions were observed mainly in the Golgi complex and vesicles of the parasympathetic ganglion nucleus of the superior cervical ganglion. Zinc ions may participate in the synthesis and assembly of some zinc containing proteins in the neurons, and maintain the three structure of some proteins. Zinc ions in the spinal ganglia and the sciatic nerve are also widely distributed. The mechanism and function of the accumulation and transport of zinc ions in the peripheral nervous system neurons is not clear. The effect of the axon transport of zinc ions in the sympathetic trunk and the effect of zinc deficiency on the structure of sympathetic dry nerve fibers in this study was studied in detail.
Materials and methods
1, Wistar rats, weighing about 150g, were randomly divided into 3 groups.
(1) the first group was performed by unilateral sympathetic trunk clamp, and the side was not operated as the control. After the operation, it survived 2,4,6 hours, and then injected sodium selenide, then perfusion, and used metal autography (AMG) to observe the transport of the axon in the sympathetic trunk.
(2) the second groups were treated with the same surgical method as above, and the distribution of zinc ions in sympathetic trunk was observed by AMG electron microscopy.
(3) the third groups of rats were clamped on the unilateral sympathetic trunk, injected with sodium selenide near the clamp point, and the other side was not clamped as the control. After 24h, the upper cervical ganglia were taken down by AMG staining, and the retrograde transport of zinc ions in the sympathetic trunk was observed.
2, 3 weeks after birth, CD-1 mice were randomly divided into normal group and zinc deficiency group (ZnD) group. They were fed with normal feed and zinc deficiency diet. After 5 weeks, the sympathetic trunk was perfused. The structural changes of sympathetic trunk were observed by conventional electron microscopy, and the number of myelinated and unmyelinated nerve fibers were compared under light microscope, and the number of two groups was compared with t test. According to the difference between them.
experimental result
1, after the sympathetic trunk clamp, the results of the AMG experiment can see the accumulation of AMG positive particles at the distal and proximal end of the two clamp points. This particle is mainly located near the clamp point, but there is no positive particles between the two clamp points, and the AMG positive particles increase with the prolongation of the survival time of the rat after hand operation.
2, AMG electron microscopy showed that there were AMG positive products of zinc ions in unmyelinated nerve fibers.
3, in the AMG experimental results of the sympathetic dry retrograde transport, only about 5% of the neurons in the superior cervical ganglia were found to have zinc positive reaction products, scattered in the cytoplasm of the neurons, with deep staining around the nucleus and no positive reaction in the nucleus, and some linear AMG positive particles were found in some neurites.
4, after zinc deficiency, the ultrastructural changes in the sympathetic trunk of mice were changed, showing myelin degeneration, irregular arrangement, unclear boundary, the collapse of the nerve fiber bundle membrane, the decrease of the transverse area of the axon, and the increase in the number of abnormal nerve fibers. Increase.
discuss
It was reported that after the sciatic nerve clamp, the AMG positive reaction particles were observed at both ends of the clamp point by AMG staining, indicating the accumulation of zinc ions in the distal and proximal ends of the sciatic nerve clamp point, suggesting that the axon transport of zinc ions exists in the sciatic nerve and the ultrastructure of the sciatic nerve under the condition of zinc deficiency under the electron microscope is observed under electron microscope. It was found that the number and surface area of the myelinated nerve fibers were reduced compared to those of the normal animals, and the destruction of the myelin sheath became irregular and denatured. These showed that zinc might have a certain effect on the related functions of the peripheral nerves. Therefore, we speculate that there is also the transport of zinc ions in the sympathetic trunk and the preservation of its structure and its structure. The function plays a key role. Using AMG and AMG electron microscopy, this experiment confirms that the free zinc ions are transported to the axon in the axon of the sympathetic trunk. It is found that after the clamp operation, the sodium selenide is injected, and the accumulation of zinc ions in different degrees on both sides of the clamp point has been accumulated, and it has a lengthening of zinc separation with the survival time after the operation. The increase of the accumulation of subcellular accumulation suggests that most or all zinc ions in the zinc containing neurons in the post ganglion cells may be transported to the end of the axon and regulate some physiological functions by transport, and zinc ions are transported from the end of the axon to the cell. AMG technology is used to detect the zinc ion in the sympathetic trunk injection of sodium selenate 24h. The expression of zinc containing neurons in the superior cervical ganglion showed that about 5% neurons in the upper cervical ganglia had AMG positive reaction products, which further confirmed the retrograde axonal transport of zinc ions in the sympathetic trunk.
conclusion
1, sympathetic trunk has the anterograde and retrograde transport of zinc ions. The homogeneous AMG positive reaction can be seen in the stained sections of rat sympathetic trunk after operation.
2, the changes in sympathetic stem structure and quantity after zinc deficiency suggest that zinc ions may play an important role in maintaining the structure and function of peripheral nerve fibers.

【学位授予单位】：中国医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R322.85;R341;R361

【参考文献】