本文關鍵詞： 膠質細胞源性神經營養(yǎng)因子 背根神經節(jié) 神經元 P物質　出處：《山東大學》2008年碩士論文　論文類型：學位論文

【摘要】： 膠質細胞源性神經營養(yǎng)因子(glial cell line derived neurotrophic factor,GDNF)是轉化生長因子-β家族的成員,被認為是一種在體內有廣泛表達并且作用復雜的多效能神經營養(yǎng)因子,目前已知在離體培養(yǎng)的情況下,GDNF對周圍神經系統(tǒng)許多部分神經元具有明顯的營養(yǎng)活性。 GDNF對受刺激或損傷的背根神經節(jié)(dorsal root ganglion,DRG)神經元結構及功能的可塑性具有影響,GDNF能夠介導DRG神經元的細胞內事件,如神經元突起內線粒體的運輸或聚集以及細胞內鈣離子濃度的變化等。但GDNF對正常DRG神經元有沒有特異性作用尚不清楚。 辣椒素可以激活感覺神經元而引起痛覺,并呈劑量相關性地釋放P物質(substance P,SP)和降鈣素基因相關肽(calcitonin gene-related peptide,CGRP)等神經肽類物質和興奮性氨基酸。辣椒素受體(vanilloid receptor,VR1),廣泛分布于DRG神經元上,可被多種傷害刺激激活,是傷害性感受器的興奮因子。 SP是一種興奮性神經遞質,分布于中樞和外周神經系統(tǒng)。SP在DRG小型神經元胞體合成后,經快速軸漿運輸至中樞端及外周端,而神經遞質釋放量的多少反映了神經元的功能狀態(tài),神經遞質的表達與神經元的功能狀態(tài)有關,其釋放量的多少反映了神經元的功能狀態(tài)。小劑量應用辣椒素可興奮感覺神經末梢,并引起感覺神經遞質的釋放,但是GDNF對培養(yǎng)的DRG神經元功能狀態(tài),如神經遞質SP的釋放的影響作用目前尚不清楚。 基于以上研究背景,本研究將原代分散培養(yǎng)的胎鼠DRG神經元施加不同濃度GDNF,探測GDNF對培養(yǎng)的胎鼠DRG神經元中SP釋放的影響作用。將胎齡為15d的Wistar胎鼠DRG神經元分散培養(yǎng),觀察有GDNF(5ng/ml,10ng/ml,50ng/ml)和沒有GDNF孵育的神經元的活細胞生長狀況,用微管相關蛋白2(microtubule associated protein 2,MAP2)和4′,6二脒基-2-苯吲哚(4′,6-Diamidino-2-phenylindole,DAPI)雙染DRG神經元,計數(shù)神經元數(shù)目,并用放射免疫分析法(radioimmunoassay,RIA)檢測辣椒素孵育前、后SP的釋放,用Western blot分析SP和VR1的表達。根據(jù)以上各項檢測結果綜合分析GDNF對DRG神經元SP釋放的影響作用及其機制。 實驗結果如下: (1)各組DRG神經元倒置相差顯微鏡動態(tài)觀察結果:正常培養(yǎng)的DRG神經元呈單層散在分布,由單個神經元發(fā)出的突起相互交織成網狀;用GDNF孵育的神經元用GDNF孵育的神經元生長狀況良好。 (2)MAP2和DAPI熒光雙染:可見MAP2標記的神經元和DAPI標記的細胞核,隨著GDNF孵育濃度的增加神經元胞體更加密集。 (3)神經元計數(shù)結果:用GDNF孵育的標本,單位視野內神經元的數(shù)目明顯增加,與對照組相比具有顯著性差異(P＜0.05)。 (4)SP釋放量檢測結果:用GDNF孵育的標本,SP的基礎釋放量和由辣椒素刺激后誘發(fā)的SP釋放量均較對照組顯著性增加,而且由辣椒素刺激后誘發(fā)的SP釋放量增加的幅度明顯高于SP基礎釋放量增加的幅度。由辣椒素刺激后誘發(fā)的SP釋放量與GDNF呈劑量依賴關系。 (5)Western blot分析SP表達的結果:與對照組相比,GDNF孵育的標本,SP表達量增加。 (6)Western blot分析VR1表達的結果:與對照組相比,GDNF孵育的標本,VR1表達量增加。 以上結果提示:GDNF可促進培養(yǎng)的胎鼠DRG神經元的存活,能促進培養(yǎng)中DRG神經元神經遞質SP的表達及增加SP的基礎釋放量,并能增加辣椒素誘發(fā)神經遞質SP釋放的敏感性。
[Abstract]:Glial cell line derived neurotrophic factor ( GDNF ) is a member of transforming growth factor - 尾 family , which is considered to be a potent neurotrophic factor which is widely expressed in vivo and has a complex role . GDNF has an influence on the structure and function of dorsal root ganglion ( DRG ) neurons stimulated or injured , and GDNF can mediate intracellular events of DRG neurons , such as the transport or aggregation of mitochondria in the neurons and the changes of intracellular calcium ion concentration . However , the specific effect of GDNF on normal DRG neurons is unclear . Capsaicin can activate sensory neurons to cause pain , and release neuropeptides such as substance P ( SP ) and calcitonin gene - related peptide ( CGRP ) in dose - related manner . SP is an excitatory neurotransmitter , distributed in that central and peripheral nervous system . The effects of GDNF on SP release in cultured fetal rat DRG neurons were investigated . The effects of GDNF ( 5 ng / ml , 10 ng / ml , 50 ng / ml ) and GDNF - free cultured neurons were observed . The effects of GDNF on SP release in DRG neurons were analyzed by radioimmunoassay ( RIA ) . The effects of GDNF on SP release of DRG neurons were analyzed by radioimmunoassay ( RIA ) . The experimental results are as follows : ( 1 ) The results of dynamic observation of the inverted phase contrast microscope of DRG neurons in each group : the DRG neurons cultured normally were scattered in a single layer , the protrusions emanating from the individual neurons interweave into a net mesh , and the neurons incubated with GDNF were in good condition with GDNF - incubated neurons . ( 2 ) Both MAP2 and DAPI fluorescent dyes showed that MAP2 - labeled neurons and DAPI - labeled nuclei were more dense with the increase of GDNF incubation concentration . ( 3 ) The results of neuron counting : the number of neurons in the unit field of view was significantly increased compared with the control group ( P < 0.05 ) . ( 4 ) The results of SP release showed that the basal release of SP and the release of SP induced by capsaicin were significantly higher than those in control group , and the amplitude of SP release induced by capsaicin was significantly higher than that in control group . The amount of SP release induced by capsaicin was dose - dependent . ( 5 ) Western blot analysis of SP expression showed that the expression of SP increased compared with the control group . ( 6 ) The results of Western blot analysis showed that the expression of GDNF increased in the samples incubated with GDNF as compared with the control group . These results suggest that GDNF can promote the survival of cultured rat DRG neurons , promote the expression of the neurotransmitter SP in cultured DRG neurons and increase the basal release amount of SP , and increase the sensitivity of capsaicin - induced release of SP .

【學位授予單位】：山東大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R33

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相關期刊論文 前4條

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