本文選題：鋅離子 + 交感干��； 參考：《中國醫(yī)科大學》2008年碩士論文

【摘要】： 前言 鋅是人體內重要的微量元素之一,是正常生長發(fā)育、基因表達、蛋白質代謝、免疫功能等過程中所必需的元素。近年來,人們對鋅與神經(jīng)系統(tǒng)生長發(fā)育以及功能調節(jié)之間的關系進行了廣泛的研究,發(fā)現(xiàn)鋅缺乏可以引起神經(jīng)系統(tǒng)多種疾病的發(fā)生。在中樞神經(jīng)系統(tǒng)中,大部分鋅離子與蛋白質結合并維持蛋白質分子的三級結構,小部分的鋅離子以游離方式存在于含鋅神經(jīng)元(zinc-enriched neuron,ZEN)軸突終末的突觸小泡中。在突觸活動中鋅離子與谷氨酸、γ-氨基丁酸(gamma-aminobutyric acid,GABA)等神經(jīng)遞質共同釋放到突觸間隙,參與學習和記憶的形成、神經(jīng)系統(tǒng)發(fā)育以及運動和感覺功能的調節(jié)。鋅離子也存在于周圍神經(jīng)系統(tǒng)中,有文獻報道幾乎所有的頸上神經(jīng)節(jié)(superior cervical ganglia,SCG)神經(jīng)元中均有鋅離子分布,電鏡觀察游離鋅離子主要聚集在頸上神經(jīng)節(jié)交感節(jié)后神經(jīng)元核周的高爾基復合體和囊泡內,鋅離子可能參與了神經(jīng)元內某些含鋅蛋白質的合成與組裝,并維持某些蛋白質的三級結構。在脊神經(jīng)節(jié)以及坐骨神經(jīng)中鋅離子也有廣泛分布。然而,鋅離子在周圍神經(jīng)系統(tǒng)神經(jīng)元內的聚集轉運機制以及功能尚不清楚。本研究對鋅離子在交感干內的軸突運輸以及鋅缺乏對交感干神經(jīng)纖維結構的影響進行了詳細的研究。 材料與方法 1、Wistar大鼠,體重約150g,雌雄不限,隨機分成3組。 (1)第一組進行單側交感干鉗夾手術,對側未做手術作為對照,分別使其術后存活2、4、6小時,在此期間注射硒化鈉,然后灌流取材,采用金屬自顯影(AMG)的方法觀察鋅離子在交感干中的軸突運輸情況。 (2)第二組采用與上述同樣的手術方法,灌流取材后應用AMG電鏡的方法觀察鋅離子在交感干中的分布情況。 (3)第三組大鼠鉗夾單側交感干,在靠近鉗夾點的上方注射硒化鈉,同樣另一側未鉗夾作為對照,24h后灌流取下兩側頸上神經(jīng)節(jié)進行AMG染色,觀察交感干中鋅離子的逆行運輸。 2、生后3周的CD-1小鼠隨機分為正常組和缺鋅組(ZnD)組,分別給予正常飼料和缺鋅飼料喂養(yǎng),5周后灌流取交感干,應用常規(guī)電鏡技術觀察交感干的結構改變,并在光鏡下比較有髓神經(jīng)纖維和無髓神經(jīng)纖維的數(shù)量變化,應用t檢驗比較兩組數(shù)據(jù)之間的差異。 實驗結果 1、交感干鉗夾后,AMG實驗結果中可以看到在兩個鉗夾點的遠端和近端有AMG陽性顆粒的蓄積,此顆粒主要位于靠近鉗夾點處,但是在兩個鉗夾點之間沒有陽性顆粒的存在,并且AMG陽性顆粒隨著手術后大鼠存活時間的延長而增多。 2、AMG電鏡結果顯示在無髓神經(jīng)纖維中有鋅離子的AMG陽性反應產(chǎn)物。 3、在交感干逆行運輸?shù)腁MG實驗結果中,光鏡下觀察頸上神經(jīng)節(jié)中僅有約5%的神經(jīng)元有鋅離子陽性反應產(chǎn)物,彌散分布于神經(jīng)元的胞質內,細胞核周圍染色較深,細胞核未見陽性反應,一些神經(jīng)元突起內可見點線狀的AMG陽性顆粒。 4、鋅缺乏后導致小鼠交感干超微結構的改變,表現(xiàn)為髓鞘變性、排列不規(guī)則、界限不清楚,神經(jīng)纖維束膜萎陷,神經(jīng)軸突的橫截面積減小,形態(tài)異常的神經(jīng)纖維數(shù)量增多。光鏡下觀察,缺鋅小鼠交感干有髓神經(jīng)纖維的數(shù)量減少,無髓神經(jīng)纖維增多。 討論 有文獻報道,坐骨神經(jīng)鉗夾后通過AMG染色方法觀察到AMG陽性反應顆粒存在于鉗夾點兩端,說明坐骨神經(jīng)鉗夾點的遠側端和近側端有鋅離子的蓄積,提示在坐骨神經(jīng)中存在鋅離子的軸突運輸,電鏡下觀察缺鋅情況下的坐骨神經(jīng)超微結構,發(fā)現(xiàn)有髓神經(jīng)纖維的數(shù)量和表面積與正常飼養(yǎng)的動物相比都有所減少,髓鞘破壞變得不規(guī)則,并且發(fā)生變性,這些都證明鋅可能對周圍神經(jīng)的相關功能具有一定的作用,因而我們推測交感干中也有鋅離子的運輸,并且對其結構的保持及功能的發(fā)揮起到關鍵性作用。應用AMG和AMG電鏡技術,本實驗證實了游離鋅離子在交感干的軸突內進行軸突運輸。研究發(fā)現(xiàn),鉗夾手術后,注射硒化鈉,在鉗夾點的兩側端均有不同程度的鋅離子蓄積,并且有隨著手術后存活時間的延長鋅離子蓄積增多的現(xiàn)象,提示大多數(shù)或全部交感節(jié)后含鋅神經(jīng)元胞體內的鋅離子可能通過順行運輸?shù)竭_軸突終末并參與某些生理功能的調節(jié),而且也有鋅離子由軸突終末向胞體逆行運輸。應用AMG技術檢測交感干注射硒酸鈉24h后,鋅離子在頸上神經(jīng)節(jié)中含鋅神經(jīng)元的表達,光鏡下結果顯示,頸上神經(jīng)節(jié)中約有5%神經(jīng)元有AMG陽性反應產(chǎn)物,此結果進一步證實了鋅離子在交感干中進行逆行的軸突運輸。 結論 1、交感干存在著鋅離子的順行和逆行運輸,在手術后的大鼠交感干的染色切片中可以看到均勻的AMG陽性反應。 2、缺鋅后交感干結構和數(shù)量的變化說明鋅離子可能對周圍神經(jīng)纖維結構的保持及功能的發(fā)揮起到重要的作用。
[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.

【學位授予單位】：中國醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R322.85;R341;R361

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