GDNF对培养的背根神经节神经元P物质释放的影响及作用机制

发布时间：2018-01-29 10:14

本文关键词： 胶质细胞源性神经营养因子背根神经节神经元 P物质　出处：《山东大学》2008年硕士论文　论文类型：学位论文

【摘要】： 胶质细胞源性神经营养因子(glial cell line derived neurotrophic factor,GDNF)是转化生长因子-β家族的成员,被认为是一种在体内有广泛表达并且作用复杂的多效能神经营养因子,目前已知在离体培养的情况下,GDNF对周围神经系统许多部分神经元具有明显的营养活性。 GDNF对受刺激或损伤的背根神经节(dorsal root ganglion,DRG)神经元结构及功能的可塑性具有影响,GDNF能够介导DRG神经元的细胞内事件,如神经元突起内线粒体的运输或聚集以及细胞内钙离子浓度的变化等。但GDNF对正常DRG神经元有没有特异性作用尚不清楚。辣椒素可以激活感觉神经元而引起痛觉,并呈剂量相关性地释放P物质(substance P,SP)和降钙素基因相关肽(calcitonin gene-related peptide,CGRP)等神经肽类物质和兴奋性氨基酸。辣椒素受体(vanilloid receptor,VR1),广泛分布于DRG神经元上,可被多种伤害刺激激活,是伤害性感受器的兴奋因子。 SP是一种兴奋性神经递质,分布于中枢和外周神经系统。SP在DRG小型神经元胞体合成后,经快速轴浆运输至中枢端及外周端,而神经递质释放量的多少反映了神经元的功能状态,神经递质的表达与神经元的功能状态有关,其释放量的多少反映了神经元的功能状态。小剂量应用辣椒素可兴奋感觉神经末梢,并引起感觉神经递质的释放,但是GDNF对培养的DRG神经元功能状态,如神经递质SP的释放的影响作用目前尚不清楚。基于以上研究背景,本研究将原代分散培养的胎鼠DRG神经元施加不同浓度GDNF,探测GDNF对培养的胎鼠DRG神经元中SP释放的影响作用。将胎龄为15d的Wistar胎鼠DRG神经元分散培养,观察有GDNF(5ng/ml,10ng/ml,50ng/ml)和没有GDNF孵育的神经元的活细胞生长状况,用微管相关蛋白2(microtubule associated protein 2,MAP2)和4′,6二脒基-2-苯吲哚(4′,6-Diamidino-2-phenylindole,DAPI)双染DRG神经元,计数神经元数目,并用放射免疫分析法(radioimmunoassay,RIA)检测辣椒素孵育前、后SP的释放,用Western blot分析SP和VR1的表达。根据以上各项检测结果综合分析GDNF对DRG神经元SP释放的影响作用及其机制。实验结果如下: (1)各组DRG神经元倒置相差显微镜动态观察结果:正常培养的DRG神经元呈单层散在分布,由单个神经元发出的突起相互交织成网状;用GDNF孵育的神经元用GDNF孵育的神经元生长状况良好。 (2)MAP2和DAPI荧光双染:可见MAP2标记的神经元和DAPI标记的细胞核,随着GDNF孵育浓度的增加神经元胞体更加密集。 (3)神经元计数结果:用GDNF孵育的标本,单位视野内神经元的数目明显增加,与对照组相比具有显著性差异(P＜0.05)。 (4)SP释放量检测结果:用GDNF孵育的标本,SP的基础释放量和由辣椒素刺激后诱发的SP释放量均较对照组显著性增加,而且由辣椒素刺激后诱发的SP释放量增加的幅度明显高于SP基础释放量增加的幅度。由辣椒素刺激后诱发的SP释放量与GDNF呈剂量依赖关系。 (5)Western blot分析SP表达的结果:与对照组相比,GDNF孵育的标本,SP表达量增加。 (6)Western blot分析VR1表达的结果:与对照组相比,GDNF孵育的标本,VR1表达量增加。以上结果提示:GDNF可促进培养的胎鼠DRG神经元的存活,能促进培养中DRG神经元神经递质SP的表达及增加SP的基础释放量,并能增加辣椒素诱发神经递质SP释放的敏感性。
[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 .

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R33

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