谷氨酸信号通路调控恶性黑素瘤侵袭与增殖的实验研究

摘要/Abstract

摘要：

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100 μl的PBS液；②MK801组加入100 μmol/L NMDAR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。

关键词: 谷氨酸信号通路恶性黑素瘤

Abstract:

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, 0.01 and 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.

Key words: glutamate signaling pathway ,melanoma

李丽丽单路娟张媛高船舟高海芹高文婷刘越坚宋智琦. 谷氨酸信号通路调控恶性黑素瘤侵袭与增殖的实验研究[J]. 中华皮肤科杂志, 2011, 44(3): 186-190.

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