CAJ | 학술논문

目的探讨谷氨酸信号通路在黑素转运中的作用.方法原代培养并纯化黑素细胞及角质形成细胞,免疫荧光显微镜观察离子型谷氨酸受体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)在黑素细胞内的分布,共聚焦显微镜观察100 μmol/L NMDAR激动剂NMDA和100μmol/L拮抗剂地卓西平马来酸盐(dizocilpine maleate,MK801)作用5 min和1h后黑素细胞内钙离子浓度的变化以及100 μmol/L MK801对黑素细胞内微管蛋白的影响.结果 100 μmol/L NMDA可使黑素细胞内瞬时钙离子浓度升高,但100μmol/L MK801可使其降低；MK801先作用于黑素细胞5 min或1h阻断NMDA受体后,NMDA均不能再次诱导瞬时钙离子浓度升高.共聚焦显微镜观察发现MK801作用24 h后,胞内微管蛋白重新分布聚集于核周.扫描电镜观察发现100 μmol/L MK801作用于黑素细胞-角质形成细胞共培养体系48 h后,黑素细胞和角质形成细胞之间以及两种细胞表面的丝状伪足数量明显减少.共培养体系下,100μmol/L MK801作用后,角质形成细胞中的黑素含量明显降低,即从黑素细胞向角质形成细胞转移的黑素数量明显减少.结论谷氨酸信号通路对黑素细胞胞内钙离子浓度、微管蛋白分布、黑素细胞伪足形成以及黑素细胞及角质形成细胞间的黑素转运具有一定调节作用.
목적 탐토곡안산신호통로재흑소전운중적작용.방법 원대배양병순화흑소세포급각질형성세포,면역형광현미경관찰리자형곡안산수체N-갑기-D-천동안산수체1(NMDAR1)화N-갑기-D-천동안산수체2A( NMDAR2A)재흑소세포내적분포,공취초현미경관찰100 μmol/L NMDAR격동제NMDA화100μmol/L길항제지탁서평마래산염(dizocilpine maleate,MK801)작용5 min화1h후흑소세포내개리자농도적변화이급100 μmol/L MK801대흑소세포내미관단백적영향.결과 100 μmol/L NMDA가사흑소세포내순시개리자농도승고,단100μmol/L MK801가사기강저；MK801선작용우흑소세포5 min혹1h조단NMDA수체후,NMDA균불능재차유도순시개리자농도승고.공취초현미경관찰발현MK801작용24 h후,포내미관단백중신분포취집우핵주.소묘전경관찰발현100 μmol/L MK801작용우흑소세포-각질형성세포공배양체계48 h후,흑소세포화각질형성세포지간이급량충세포표면적사상위족수량명현감소.공배양체계하,100μmol/L MK801작용후,각질형성세포중적흑소함량명현강저,즉종흑소세포향각질형성세포전이적흑소수량명현감소.결론 곡안산신호통로대흑소세포포내개리자농도、미관단백분포、흑소세포위족형성이급흑소세포급각질형성세포간적흑소전운구유일정조절작용.
Objective To investigate the roles of glutamate signaling pathway in melanin transfer.Methods Epidermal melanocytes and keratinocytes were isolated from human foreskin tissue followed by purification and primary culture.Immunofluorescence microscopy was conducted to observe the intracellular distribution of N-methy-D-aspartate receptor 1 (NMDAR1) and NMDAR2A in melanocytes.Some melanocytes were classified into 4 groups to be pretreated with MK801 (the NMDAR antagonist dizocilpine maleate) at 100μmol/L for 5 minutes followed by treatment with NMDA (an NMDAR agonist) at 100 μmol/L (MK801-pretreated group 1),pretreated with MK801 at 100 μmol/L for 1 hour followed by treatment with NMDA at 100μmol/L (MK801-pretreated group 2),treated with MK801 at 100 μmol/L for 5 minutes (MK801 group),treated with NMDA at 100 μmol/L for 5 minutes (NMDA group),respectively,then,confocal microscopy was performed to measure the intracellular calcium (Ca2+) concentration of the melanocytes.The distribution of β-tubulin was visualized by confocal microscopy in melanocytes treated with MK801 at 100 μmol/L for 24 hours.Some melanocytes and keratinocytes were cocultured with or without MK801 at 100 μmol/L for 24 or 48 hours,then,scaning microscopy was carried out to observe the junction structure between melanocytes and keratinocytes,and alkali method coupled with spectrophotometric analysis to determine melanin content in keratinocytes.Results The intracellular calcium concentration of melanocytes was decreased by MK-801,but increased by NMDA at 100 μmol/L,and the increase was blocked by the pretreatment with MK-801 for 5 minutes or 1 hour.After incubation with MK-801 at 100 μmol/L for 24 hours,a more intense staining for β-tubulin was observed around the nuclei of melanocytes.There was a significant reduction in the number of filopodia on the surface of and between melanocytes and keratinocytes after treatment with MK-801 at 100 μmol/L for 48 hours.Also,the content of melanin (represented as the absorbance value at 375 nm) transferred from melanocytes into keratinocytes was statistically reduced in coculture system treated with MK-801 at 100 μmol/L compared with that without treatment (0.158 ± 0.003 vs.2.203 ± 0.006,t =6.323,P ＜ 0.01 ).Conclusions The glutamate signaling pathway exerts a regulatory effect on intracellular calcium concentration of distribution of β-tubulin in,filopodia formation of melanocytes and melanin transfer between melanocytes and keratinocytes.