CAJ | 학술논문

目的探讨谷氨酸信号通路在黑素细胞及角质形成细胞伪足形成及黑素小体转运中的作用机制.方法原代培养并纯化黑素细胞、角质形成细胞,建立黑素细胞与角质形成细胞体外共培养体系.扫描电镜观察离子型谷氨酸受体N-甲基-D-天冬氨酸(NMDA)受体非竞争性拮抗剂MK801及激动剂NMDA作用下黑素细胞、角质形成细胞的伪足形态变化,以311 nm中波紫外线(UVB)照射为阳性对照.免疫荧光双染色激光共聚焦显微镜下观察MK801及NMDA对黑素小体转运的调节.结果扫描电镜观察发现,100 μmol/LMK801作用24 h后黑素细胞树突末端明显变细,树突变长,树突数量无明显变化而细胞表面的丝状伪足数量明显变少,且伪足长度变短;100 μmol/L NMDA作用24 h后黑素细胞树突的末端明显变宽、树突变短,树突数量无明显变化而细胞表面丝状伪足数量明显增多,丝状伪足长度增加.黑素细胞与角质形成细胞共培养体系中,未用药组黑素细胞与角质形成细胞间有丝状伪足相连接,黑素细胞的角质形成细胞侧丝状伪足数量多于对侧.100 μmol/L MK801作用于共培养体系24 h后,黑素细胞与角质形成细胞之间的丝状伪足数量及黑素细胞伸向角质形成细胞的丝状伪足数量减少;100 μmol/L NMDA作用于共培养体系24 h后,黑素细胞与角质形成细胞之间的丝状伪足数量及黑素细胞伸向角质形成细胞的丝状伪足数量增多.激光共聚焦显微镜观察显示,共培养体系下,未用药组角质形成细胞中存在黑素小体;100 μmol/L MK801作用24h后角质形成细胞中的黑素小体数量减少;100 μmol/L NMDA作用24 h后角质形成细胞中的黑素小体数量增多,且与黑素细胞不相邻的角质形成细胞中也发现黑素小体存在.结论谷氨酸信号通路可能通过调节黑素细胞树突的形态及丝状伪足的形成参与调节黑素小体自黑素细胞至角质形成细胞的转运. 目的探討穀氨痠信號通路在黑素細胞及角質形成細胞偽足形成及黑素小體轉運中的作用機製.方法原代培養併純化黑素細胞、角質形成細胞,建立黑素細胞與角質形成細胞體外共培養體繫.掃描電鏡觀察離子型穀氨痠受體N-甲基-D-天鼕氨痠(NMDA)受體非競爭性拮抗劑MK801及激動劑NMDA作用下黑素細胞、角質形成細胞的偽足形態變化,以311 nm中波紫外線(UVB)照射為暘性對照.免疫熒光雙染色激光共聚焦顯微鏡下觀察MK801及NMDA對黑素小體轉運的調節.結果掃描電鏡觀察髮現,100 μmol/LMK801作用24 h後黑素細胞樹突末耑明顯變細,樹突變長,樹突數量無明顯變化而細胞錶麵的絲狀偽足數量明顯變少,且偽足長度變短;100 μmol/L NMDA作用24 h後黑素細胞樹突的末耑明顯變寬、樹突變短,樹突數量無明顯變化而細胞錶麵絲狀偽足數量明顯增多,絲狀偽足長度增加.黑素細胞與角質形成細胞共培養體繫中,未用藥組黑素細胞與角質形成細胞間有絲狀偽足相連接,黑素細胞的角質形成細胞側絲狀偽足數量多于對側.100 μmol/L MK801作用于共培養體繫24 h後,黑素細胞與角質形成細胞之間的絲狀偽足數量及黑素細胞伸嚮角質形成細胞的絲狀偽足數量減少;100 μmol/L NMDA作用于共培養體繫24 h後,黑素細胞與角質形成細胞之間的絲狀偽足數量及黑素細胞伸嚮角質形成細胞的絲狀偽足數量增多.激光共聚焦顯微鏡觀察顯示,共培養體繫下,未用藥組角質形成細胞中存在黑素小體;100 μmol/L MK801作用24h後角質形成細胞中的黑素小體數量減少;100 μmol/L NMDA作用24 h後角質形成細胞中的黑素小體數量增多,且與黑素細胞不相鄰的角質形成細胞中也髮現黑素小體存在.結論穀氨痠信號通路可能通過調節黑素細胞樹突的形態及絲狀偽足的形成參與調節黑素小體自黑素細胞至角質形成細胞的轉運.
목적 탐토곡안산신호통로재흑소세포급각질형성세포위족형성급흑소소체전운중적작용궤제.방법 원대배양병순화흑소세포、각질형성세포,건립흑소세포여각질형성세포체외공배양체계.소묘전경관찰리자형곡안산수체N-갑기-D-천동안산(NMDA)수체비경쟁성길항제MK801급격동제NMDA작용하흑소세포、각질형성세포적위족형태변화,이311 nm중파자외선(UVB)조사위양성대조.면역형광쌍염색격광공취초현미경하관찰MK801급NMDA대흑소소체전운적조절.결과 소묘전경관찰발현,100 μmol/LMK801작용24 h후흑소세포수돌말단명현변세,수돌변장,수돌수량무명현변화이세포표면적사상위족수량명현변소,차위족장도변단;100 μmol/L NMDA작용24 h후흑소세포수돌적말단명현변관、수돌변단,수돌수량무명현변화이세포표면사상위족수량명현증다,사상위족장도증가.흑소세포여각질형성세포공배양체계중,미용약조흑소세포여각질형성세포간유사상위족상련접,흑소세포적각질형성세포측사상위족수량다우대측.100 μmol/L MK801작용우공배양체계24 h후,흑소세포여각질형성세포지간적사상위족수량급흑소세포신향각질형성세포적사상위족수량감소;100 μmol/L NMDA작용우공배양체계24 h후,흑소세포여각질형성세포지간적사상위족수량급흑소세포신향각질형성세포적사상위족수량증다.격광공취초현미경관찰현시,공배양체계하,미용약조각질형성세포중존재흑소소체;100 μmol/L MK801작용24h후각질형성세포중적흑소소체수량감소;100 μmol/L NMDA작용24 h후각질형성세포중적흑소소체수량증다,차여흑소세포불상린적각질형성세포중야발현흑소소체존재.결론 곡안산신호통로가능통과조절흑소세포수돌적형태급사상위족적형성삼여조절흑소소체자흑소세포지각질형성세포적전운.
Objective To investigate the regulatory effect of glutamate signaling pathway on filopodia formation in epidermal cells and on melanosome transfer.Methods Epidermal melanocytes and keratinocytes were isolated from human foreskin and subjected to subculture.After two to three passages of subculture,the melanocytes and keratinocytes were cultured alone or in combination with or without the presence of MK801 (an antagonist of N-methyl-D-aspartic acid (NMDA) receptor) of 100 μmol/L,or NMDA (the activator of NMDA receptor) of 100 μmol/L,for 24 hours.The melanocytes irradiated with UVB at 311 nm served as the control.Scanning electron microscopy was used to observe the appearance of filopodia and dendrites of melanocytes and keratinocytes.Melanosome transfer was visualized under confocal laser scanning microscopy after double immunofluorescent staining.Results Although no obvious changes were observed in the number of dendrites in monocultured melanocytes after treatment with MK801 or NMDA for 24 hours,dendrites became thinner at the terminus and longer with a decrease in the number and length of filopodia after MK801 treatment,but thicker and shorter with an increase in the number and length of filopodia after NMDA treatment compared with untreated monocultured melanocytes.In the coculture system,filopodia were observed between the untreated melanocytes and keratinocytes,and the number of filopodia in melanocytes was larger in the side adjacent to keratinocytes than in the opposite side.Compared with the untreated coculture system,the number of both filopodia connecting melanocytes and keratinocytes and filopodia extending from melanocytes to keratinocytes decreased in the coculture system after treatment with MK801 of 100 μmol/L,but increased after treatment with NMDA of 100 μmol/L,for 24 hours.Melanosomes were found in keratinocytes cocultured with melanocytes without treatment,which were decreased in number after 24-hour treatment with MK801 of 100 μmol/L,but increased in number and even present in keratinocytes nonadiacent to melanocytes after 24-hour treatment with NMDA of 100 μmol/L.Conclusion Glutamate signaling pathway may modulate the transfer of melanosomes from melanocytes to keratinocytes via modulating the appearance of melanocyte dendrites and formation of filopodia.