【作者】 卢平；

【导师】 宋智琦；

【作者基本信息】 大连医科大学 ， 皮肤病与性病学， 2008， 硕士

【摘要】 目的:研究恶黑细胞中的谷氨酸信号通路及其与细胞骨架蛋白分子的作用机制。方法:1微管相关蛋白2a (Microtubule-associated protein 2a,MAP2a)、离子型谷氨酸受体中的甲基-D-天冬氨酸受体2A(N-methyl-D-aspartate receptors 2A,NMDAR2A)、代谢型谷氨酸受体1(metabotropic glutamate receptor 1,mGluR1)在人类侵袭性恶黑细胞WM451LU细胞中的亚细胞分布差异研究:培养人胚肾细胞系HEK293细胞、WM451LU细胞;在HEK293细胞中扩增重组腺病毒-微管相关蛋白2a(adenovirus-mediated MAP2a,Ad-MAP2a);对WM451LU细胞MAP2a基因转染后进行免疫荧光双重染色及倒置荧光显微镜下观察WM451LU细胞中MAP2a、NMDAR2A及mGluR1的细胞内分布;进一步在激光扫描共聚焦显微镜下观察WM451LU细胞中MAP2a与NMDAR2A及mGluR1的亚细胞分布。2上调MAP2a表达及神经保护化学药干预对WM451LU细胞形态的影响观察:观察转染Ad-MAP2a、NMDAR非竞争性拮抗剂MK-801以及选择性的非竞争性mGluR1拮抗剂CPCCOEt分别作用下,WM451LU细胞中α-微管蛋白(α-tubulin)的分布、密度以及细胞形态的变化。在原子力显微镜下观察上调MAP2a表达、MK-801及CPCCOEt分别及协同作用下WM451LU细胞树突形态的影响。3转染Ad-MAP2a对WM451LU细胞内NMDAR2A及mGluR1蛋白表达水平的影响:通过流式细胞仪及Western Blot方法测定转染Ad-MAP2a后WM451LU细胞内NMDAR2A及mGluR1的表达。4观察CPCCOEt及MK-801对WM451LU细胞生长的影响。结果:1研究发现在人类侵袭性恶黑细胞WM451LU细胞中有NMDAR2A及mGluR1的表达。NMDAR2A及mGluR1与微管相关蛋白MAP2a在WM451LU细胞中具有相同的亚细胞定位:均分布于细胞浆内,并且主要位于细胞树突内。2转染Ad-MAP2a后WM451LU细胞内α-tubulin的密度明显增加,细胞的树突明显增长,并且α-tubulin主要位于增长的细胞树突内。在谷氨酸受体拮抗剂MK-801及CPCCOEt作用下WM451LU细胞内α-tubulin的密度明显增大,细胞的树突纤细而狭长,明显不同于阴性对照组细胞短而粗的细胞树突,且α-tubulin主要分布于纤细、狭长的细胞树突内。3 MK-801单独以及与MAP2a协同作用下WM451LU细胞恢复树突化,其中以二者协同作用下细胞形态的改变更为显著,细胞树突明显延长,出现大量二级、三级细胞树突,呈现出与正常黑色素细胞所特有的细胞形态极其相似的细胞形态。4转染Ad-MAP2a后NMDAR2A及mGluR1的表达量无明显变化。5 MK-801及CPCCOEt对人类侵袭性恶黑细胞WM451LU细胞增殖具有显著抑制作用。结论:1 WM451LU细胞中有NMDAR2A及mGluR1的表达,且其亚细胞分布与MAP2a一致。2 MK-801以及CPCCOEt具有诱导WM451LU细胞树突形成的作用,并可使WM451LU细胞树突内的微管蛋白密度增加。3 MK-801单独以及与MAP2协同作用下可诱导WM451LU细胞树突形成,使其呈现出与正常黑色素细胞所特有的细胞形态极其相似的细胞形态,其中以二者协同作用下细胞形态的改变更为显著。4 MK-801以及CPCCOEt体外可抑制WM451LU细胞的增殖。更多还原

【Abstract】 Background:Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system (CNS). It acts via a number of glutamate receptor classes to induce neuronal depolarization. Glutamate receptors can be subdivided into ionotropic receptors, and the metabotropic receptors. In addition to its central function, regulatory effects of glutamate have been described in peripheral tissues such as bone, pancreas and skin. In the epidermis, glutamate is believed to play a role in barrier homeostasis and wound healing via NMDA receptors on keratinocytes. Expression of a functional mGluR5 has been reported on melanocytes, where it is not only suggested to be involved in the control of melanocyte proliferation, but also harbours potential toxicity and may ontribute to cell damage. Glutamate toxicity has been studied extensively in neurones. Excessive stimulation of both ionotropic and metabotropic glutamate receptors leads to cell death. Blockage of NMDA receptors with MK801 caused a melanocyte morphology change, which was associated with disorganisation of actin and tubulin microfilaments. Whereas the role of the glutamate signalling system in neurones is well documented, the presence of the various glutamate receptors on melanoma cells and their effects on melanoma cells behaviour has not been studied in detail. It is also unclear whether melanoma cells defects, such as in the pigmentary disorder vitiligo where functional melanocytes are lost from the epidermis by unknown mechanisms, could be related to glutamate receptors. It is necessary to elucidate the glutamate receptors expression of human melanoma cells and the effects of glutamate receptor agonists and antagonists on melanoma cells morphology and growth.Microtubule-associated proteins (MAPs) are a family of proteins that bind to and stabilize microtubules (MTs). MAP2 is one isoform of MAPs. Expression of MAP2 in vitro in heterologous cells induces rapid formation of stable MT bundles and cellular processes. Vijayasaradhi et al found that MAP2, a neuronspecific protein, is expressed abundantly in early invasive primary melanoma lesions and primary melanoma cell lines but is absent in metastatic melanomas lesions and cell lines. Investigation of the mechanisms that underlie the effect of MAP2 on melanoma progression showed that MAP2 expression in metastatic melanoma cell lines leads to microtubule stabilization, cell cycle arrest in G2-M phase and growth inhibition. Melanoma exhibits plasticity of differentiation and is known to differentiate along multiple, including endothelial and neuronal, cellular pathways. However, the effects of MAP2 on melanoma cells on morphology and growth are not well understood.Objective: Study the Glutamate signaling in melanoma cell and molecular mechanism with cytoskeleton protein.Methods:To investigate the expression, subcellular localization ,effects on melanoma growth, invasion and cell morphology change of MAP2a, NMDAR2A and mGluR1,we performed double-immunofluorescence staining coupled with confocal microscopy analysis, three dimensional atomic force microscopy analysis, flow cytometry analysis, tumor cell growth assay and in vivo study, mainly used human metastatic melanoma cell line WM451LU.Results:The results demonstrate that MAP2a, NMDAR2A and mGluR1 have similar subcellular localization, and for the first time showed that combined adenovirus-mediated MAP2a and NMDAR antagonist MK-801 treated melanoma cells led to morphologic alteration, the tumor cell represented the secondary and tertiary branches resembled normal melanocytes. NMDAR antagonist MK-801 and mGluR1 antagonist CPCCOEt showed the significant tumor growth suppression of melanomas in vitro. Therefore, MAP2a, NMDAR2A and mGluR1 appear to be a promising target for melanoma therapy.更多还原