谷氨酸信号通路在表皮中的作用研究

发布时间：2019-02-19 20:24

【摘要】：目的:探讨谷氨酸信号通路在表皮中的作用机制。方法: 1.原代培养并纯化黑素细胞(Melanocyte,MC);倒置显微镜观察MC细胞形态;免疫荧光显微镜观察MC中离子型谷氨酸受体N-甲基-D-天冬氨酸受体1(NMDAR1)和N-甲基-D-天冬氨酸受体2A(NMDAR2A)的细胞内分布;共聚焦显微镜观察谷氨酸信号通路对MC胞内钙离子浓度的影响以及MC内β-tubulin的分布。 2.原代培养并纯化角质形成细胞(Keratinocyte,KC);倒置显微镜观察KC细胞形态;免疫荧光显微镜观察KC中NMDAR1和NMDAR2A的细胞内分布;共聚焦显微镜观察谷氨酸信号通路对KC胞内钙离子浓度的影响以及KC内β-tubulin的分布;流式细胞仪测定NMDA受体拮抗剂MK-801对KC凋亡的作用。 3.建立MC和KC共培养体系;倒置显微镜观察二者共培养的形态;扫描电镜观察MK-801作用下二者相互作用的形态学改变;酶标仪测定由MC向KC转运的黑素颗粒OD475值。结果: 1.倒置显微镜下MC呈树突样生长;免疫荧光显微镜下显示黑素细胞中的NMDAR1和NMDAR2A均主要分布在细胞浆内,而NMDA2A在树突分叉处表达更多;100μM浓度的NMDA使黑素细胞瞬时钙离子浓度升高,100μM浓度MK-801使其瞬时钙离子浓度降低,MK-801事先作用于MC5min或1h阻断了NMDA受体后,NMDA均不能再次诱发瞬时钙离子浓度升高;共聚焦显微镜观察发现MK-801作用24小时后导致黑素细胞树突回缩,胞内β-tubulin重新分布聚集于核周。 2.倒置显微镜下KC为铺路石样生长;免疫荧光显微镜下显示角质细胞中的NMDAR1和NMDAR2A均主要分布在细胞浆内;100μM浓度的NMDA使角质细胞瞬时钙离子浓度升高;流式细胞仪显示100μM的MK-801孵育角质细胞48h后凋亡细胞比例较对照组无明显差异;共聚焦显微镜下发现MK-801作用24小时后导致角质细胞核周的β-tubulin密度增大。 3.扫描电镜观察发现100μM MK-801作用于共培养体系48h后,MC树突数量无明显变化,KC之间相互连接的丝状伪足的数量、KC表面的丝状伪足数量以及MC-KC间的丝状伪足减少;通过酶标仪测定共培养48h后,使用100μM MK-801孵育组与正常共培养的未处理组相比较,KC中的黑素颗粒OD475值明显降低,提示从MC向KC转运的黑素小体数量减少。结论: 1.MC内有NMDAR1及NMDAR2A的表达。 2.谷氨酸信号通路可影响MC胞内钙离子浓度。 3.谷氨酸信号通路通过作用于MC内β-tubulin而影响MC丝状伪足的数量,而后者是该通路影响MC和KC间黑素转运的结构基础。 4.KC内有NMDAR1及NMDAR2A的表达。 5.谷氨酸信号通路影响KC细胞形态及其胞内钙离子浓度,而该形态学改变与细胞凋亡无关;同时影响KC中β-tubulin分布。
[Abstract]:Objective: to investigate the mechanism of glutamate signaling pathway in epidermis. Methods: 1. Melanocytes (Melanocyte,MC) were cultured and purified, and the morphology of MC cells was observed by inverted microscope. The intracellular distribution of N-methyl-D-aspartate receptor 1 (NMDAR1) and N-methyl-D-aspartate receptor 2A (NMDAR2A) in MC were observed by immunofluorescence microscopy. The effect of glutamate signal pathway on intracellular calcium concentration of MC and the distribution of 尾-tubulin in MC were observed by confocal microscope. 2. Primary culture and purification of keratinocytes (Keratinocyte,KC), observation of morphology of KC cells by inverted microscope, observation of intracellular distribution of NMDAR1 and NMDAR2A in KC by immunofluorescence microscopy; The effect of glutamate signaling pathway on intracellular calcium concentration of KC and the distribution of 尾-tubulin in KC were observed by confocal microscopy, and the effect of MK-801, a NMDA receptor antagonist, on KC apoptosis was determined by flow cytometry. 3. The co-culture system of MC and KC was established, the morphology of coculture was observed by inverted microscope, the morphological changes of the interaction between the two were observed by scanning electron microscope, and the OD475 value of melanin particles transported from MC to KC was measured by enzyme labeling. Results: 1. NMDAR1 and NMDAR2A in melanocytes were mainly distributed in cytoplasm and NMDA2A was expressed more in dendritic bifurcation under inverted microscope. 100 渭 M concentration of NMDA increased the instantaneous calcium concentration of melanocytes, and 100 渭 M concentration of MK-801 decreased the instantaneous calcium concentration of melanocytes. NMDA could not induce the transient calcium concentration of melanocytes to increase again after blocking the NMDA receptor by MK-801 for 1 h or before MC5min. Confocal microscopy showed that after 24 hours of MK-801 treatment, melanocyte dendrites retracted and 尾-tubulin was redistributed and gathered around the nucleus. 2. The NMDAR1 and NMDAR2A in keratinocytes were mainly distributed in cytoplasm by immunofluorescence microscope, and the instantaneous calcium concentration of keratinocytes was increased by 100 渭 M NMDA. Flow cytometry showed that there was no significant difference in the percentage of apoptotic cells in keratinocytes incubated with 100 渭 M MK-801 for 48 h, and the density of 尾-tubulin in keratinocytes was increased after 24 hours of MK-801 exposure under confocal microscope. 3. SEM observation showed that the number of MC dendrites did not change significantly after treated with 100 渭 M MK-801 for 48 h, but the number of KC interlinked pseudopodia, the number of KC surface filamentous pseudopodia and the number of MC-KC were decreased. After 48 hours of co-culture, compared with the untreated group cultured with 100 渭 M MK-801, the OD475 value of melanin particles in KC decreased significantly, suggesting that the number of melanosomes transported from MC to KC was decreased. Conclusion: NMDAR1 and NMDAR2A are expressed in 1.MC. 2. Glutamate signaling pathway can affect the intracellular calcium concentration of MC. 3. Glutamate signaling pathway affects the number of MC filamentous pseudopodia by acting on 尾-tubulin in MC, which is the structural basis of the pathway affecting the transport of melanin between MC and KC. NMDAR1 and NMDAR2A were expressed in 4.KC. 5. Glutamate signaling pathway affects the morphology and intracellular calcium concentration of KC cells, but the morphological changes are not related to apoptosis and the distribution of 尾-tubulin in KC.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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