谷氨酸信号通路调控恶性黑素瘤侵袭与增殖的实验研究
곡안산신호통로조공악성흑소류침습여증식적실험연구
Glutamate-mediated signaling pathway regulates the invasion and growth of malignant melanoma
  • 万方数据
    中华皮肤科杂志 중화피부과잡지
    Chinese Journal of Dermatology
    2011年 3期 186-190 ,共5页

    李丽丽%单路娟%张媛%高船舟%高海芹%高文婷%刘越坚%宋智琦

    리려려%단로연%장원%고선주%고해근%고문정%류월견%송지기

    黑素瘤,实验性%细胞系,肿瘤%受体,亲代谢性谷氨酸盐%微管蛋白质类%细胞运动 黑素瘤,實驗性%細胞繫,腫瘤%受體,親代謝性穀氨痠鹽%微管蛋白質類%細胞運動
    흑소류,실험성%세포계,종류%수체,친대사성곡안산염%미관단백질류%세포운동
    Melanoma,experimental%Cell line,tumor%Receptors,metabotropic glutamate%Microtubule proteins%Cell movement
    目的 探讨谷氨酸信号通路在恶性黑素瘤发病中的作用机制.方法 取对数生长期恶性黑素瘤WM451LU细胞,分为6组,①阴性对照组加入100μl的PBS液;②MK801组加入100 μmol/L NM-DAR非竞争性拮抗剂MK801;③CPCCOEt组加入10 μmol/L代谢型谷氨酸受体1(mGluR1)非竞争性拮抗剂CPCCOEt;④MAP2组加入腺病毒-微管相关蛋白2a(Ad-MAP2a);⑤MK801+MAP2组加入100 μmol/L MK801和Ad-MAP2a;⑥CPCCOEt+MAP2组加入10 μmol/L CPCCOEt和Ad-MAP2a.用Western印迹观察转染Ad-MAP2a载体后WM451LU细胞中离子型谷氨酸受体甲基-D-天冬氨酸受体2A(NMDAR2A)的变化.并通过细胞迁移、侵袭实验分别研究上调MAP2表达与MK-801、CPCCOEt分别或共同作用下,肿瘤细胞的迁移及侵袭性的变化.同时进行了裸鼠恶性黑素瘤动物模型的体内研究,比较上述药物的体内抑瘤作用.结果 Western印迹发现,转染Ad-MAP2a的WM451LU细胞NMDAR2A表达降低.体外迁移实验结果显示,MAP2组发生迁移的细胞为(117.04±2.76)个/高倍视野(HP),MK801组(107.64±6.50)个/HP,CPCCOEt组(97.36±4.79)个/HP,MK801+MAP2组(43.28±3.02)个/HP,CPCCOEt+MAP2组(30.76±3.97)个/HP,与阴性对照组(152.3±5.75)个/HP比较,P值均＜0.01.体外侵袭实验结果显示,MAP2组发生侵袭的细胞数为(102.56±3.81)个/HP,MK801组(98.21±5.52)个/HP,CPCCOEt组(118.23±7.96)个/HP,与阴性对照组(178.43±8.75)个/HP相比,P值均＜0.05;MK801+MAP2组(43.89±5.08)个/HP,CPCCOEt+MAP2组(58.45±6.88)个/HP,与阴性对照组相比,P值均＜0.01.裸鼠体内研究发现,用药第15天时,MAP2组肿瘤体积为(224.02±46.19)mm3,MK801组(160.33±33.91)mm3,MK801+MAP2组(91.49±21.48)mm3,CPCCOEt组(202.30±52.37)mm3,CPCCOEt+MAP2组(111.13±69.81)mm3,与阴性对照组(342.70±60.92)mm3比较,P值均＜0.01.结论 体外阻滞恶性黑素细胞的谷氨酸信号通路可抑制恶性黑素细胞的迁移及侵袭;体内阻滞恶性黑素细胞的谷氨酸信号通路可抑制恶性黑素瘤的增殖,并可使肿瘤细胞的形态发生变化.
    목적 탐토곡안산신호통로재악성흑소류발병중적작용궤제.방법 취대수생장기악성흑소류WM451LU세포,분위6조,①음성대조조가입100μl적PBS액;②MK801조가입100 μmol/L NM-DAR비경쟁성길항제MK801;③CPCCOEt조가입10 μmol/L대사형곡안산수체1(mGluR1)비경쟁성길항제CPCCOEt;④MAP2조가입선병독-미관상관단백2a(Ad-MAP2a);⑤MK801+MAP2조가입100 μmol/L MK801화Ad-MAP2a;⑥CPCCOEt+MAP2조가입10 μmol/L CPCCOEt화Ad-MAP2a.용Western인적관찰전염Ad-MAP2a재체후WM451LU세포중리자형곡안산수체갑기-D-천동안산수체2A(NMDAR2A)적변화.병통과세포천이、침습실험분별연구상조MAP2표체여MK-801、CPCCOEt분별혹공동작용하,종류세포적천이급침습성적변화.동시진행료라서악성흑소류동물모형적체내연구,비교상술약물적체내억류작용.결과 Western인적발현,전염Ad-MAP2a적WM451LU세포NMDAR2A표체강저.체외천이실험결과현시,MAP2조발생천이적세포위(117.04±2.76)개/고배시야(HP),MK801조(107.64±6.50)개/HP,CPCCOEt조(97.36±4.79)개/HP,MK801+MAP2조(43.28±3.02)개/HP,CPCCOEt+MAP2조(30.76±3.97)개/HP,여음성대조조(152.3±5.75)개/HP비교,P치균＜0.01.체외침습실험결과현시,MAP2조발생침습적세포수위(102.56±3.81)개/HP,MK801조(98.21±5.52)개/HP,CPCCOEt조(118.23±7.96)개/HP,여음성대조조(178.43±8.75)개/HP상비,P치균＜0.05;MK801+MAP2조(43.89±5.08)개/HP,CPCCOEt+MAP2조(58.45±6.88)개/HP,여음성대조조상비,P치균＜0.01.라서체내연구발현,용약제15천시,MAP2조종류체적위(224.02±46.19)mm3,MK801조(160.33±33.91)mm3,MK801+MAP2조(91.49±21.48)mm3,CPCCOEt조(202.30±52.37)mm3,CPCCOEt+MAP2조(111.13±69.81)mm3,여음성대조조(342.70±60.92)mm3비교,P치균＜0.01.결론 체외조체악성흑소세포적곡안산신호통로가억제악성흑소세포적천이급침습;체내조체악성흑소세포적곡안산신호통로가억제악성흑소류적증식,병가사종류세포적형태발생변화.
    Objective To investigate the action mechanism of glutamate-mediated signaling pathway in malignant melanoma. Methods WM451LU melanoma cells in log phase were classified into 6 groups, negative control group treated with PBS (100 μl), MK801 group treated with the non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 (100 μmol/L), CPCCOEt group treated with non-competitive metabotropic glutamate receptor 1 (mGluR1) antagonist CPCCOEt, MAP2 group transfected with adenovirus vector containing microtubule associated protein 2a (Ad-MAP2a), MK801 + MAP2 group treated with MK801 of 100 μmol/L and transfected with Ad-MAP2a, CPCCOEt + MAP2 group treated with CPCCOEt of 10 μmol/L and transfected with Ad-MAP2a. Western blot was performed to detect the expression of an ionotropic glutamate receptor, i.e., N-methyl-D-aspartate receptor type 2A (NMDAR2A) in WM451LU cells transfected with Ad-MAP2a. Scratch motility assay and cell invasion assay were conducted in vitro to detect the changes in migration and invasion ability of WM451LU cells after treated with Ad-MAP2a, MK-801, CPCCOEt alone or in combination. In vivo study was carried out to compare the inhibitory effect of the above treatments on melanoma. Results Western blot revealed a decrease in the expression of NMDAR2A in WM451LU cells after transfected with Ad-MAP2a. The scratch motility assay showed that the number of migrating cells per high power field was 117.04 ± 2.76 in MAP2 group,107.64 ± 6.50 in MK801 group,97.36 ± 4.79 in CPCCOEt group, 43.28 ± 3.02 in MK801 + MAP2 group,30.76 ± 3.97 in CPCCOEt + MAP2 group,significantly different from that in the negative control group (152.3 ± 5.75,all P ＜ 0.01 ). Cell invasion assay demonstrated that the average number of invading cells per high power field in the negative control was significantly higher than that in MAP2 group, MK801 group, CPCCOEt group, MK801+MAP2 group and CPCCOEt + MAP2 group (170.43 ±8.72 vs. 98.26 ± 3.84, 97.22 ± 5.54, 112.23 ± 7.21, 42.89 ± 5.06, 58.25 ± 6.68, P ＜ 0.05, 0.05, 0.05, 0.01and 0.01, respectively).A significant decrease was observed in the average volume of experimental melanoma in mice of MAP2 group, MK801 group, MK801 + MAP2 group, CPCCOEt group and CPCCOEt + MAP2 group compared with the negative control group (224.02 ± 46.19 mm3, 160.33 ± 33.91 mm3, 91.49 ± 21.48 mm3,202.30 ± 52.37 mm3, 111.13 ± 69.81 mm3 vs. 342.70 ± 60.92 mm3, all P ＜ 0.01 ). Conclusions To block the glutamate signaling pathway in vitro can inhibit the invasion and migration of melanoma cells, and to block the pathway in vivo can inhibit the growth of malignant melanoma and alter the morphology of melanoma cells.
    원문보기 원문다운로드 사이트로이동
저자의 최근 논문