目的  研究 MTX 对 RAS 蛋白定位改变以及下游信号途径的影响，并探讨 MTX 抑制人胶质母细胞瘤细胞株 U87 生长的可能机制。方法  不同浓度的MTX（75nmol/L、375nmol/L、750nmol/L、7,500nmol/L、15,000nmol/L）处理 U87细胞后， 采用MTT 法和FCM 法分别检测细胞活力，细胞凋亡率及细胞周期。RAS-GFP 和蛋白质印迹法分别检测RAS 蛋白的分布 定位和其下游MAPK/ERK 蛋白的表达水平。采用逆转录病毒法将RAS-GFP 融合基因转导至U87 细胞，共聚焦显微镜技 术能检测到RAS 活化后定位分布，与不同的MAKP/ERK 信号途径特异性抑制剂（U0126 和 PD98059）共培养，采用蛋白 质印迹法检测MTX 对 P-P42/P44 MARK 蛋白磷酸化水平的影响以及其调控转录因子c-MYC 表达水平发的影响。50nmol/L 的 MTX 处理 U87 细胞后，进一步用 FCM 法检测细胞表面分子 CD47 的表达水平。结果  不同浓度的 MTX（75nmol/L、 375nmol/L、750nmol/L、7,500nmol/L、15,000nmol/L）均能抑制人神经胶质母细胞瘤细胞株 U87 的增殖，呈剂量依赖性， 不同浓度的MTX 可将U87 细胞阻滞于个G2/S 期，并不能直接诱导细胞发生凋亡。转导RAS-GFP 融合基因到U87 细胞 （100% GFP），同对照组相比，用MTX 处理后发现RAS 蛋白从细胞膜转移到细胞质，从而通过降低或者抑制P-p42/p44 MARK 蛋白磷酸化水平（P ＜ 0.05），以及抑制转录因子 c-MYC 的表达水平（P ＜ 0.05），导致 U87 细胞生长受到抑制。 我们还近一步证明了 MTX 抑制与神经胶质母细胞瘤生长密切相关的细胞表面分子 CD47 在不同时间的表达水平。结论  我 们首次证明MTX通过改变RAS蛋白定位抑制MAPK/ERK/C-MYC信号导致神经胶质母细胞瘤U87细胞株的生长收到抑制， 细胞停滞在 G2/S 期，提示 MTX 有可能作为复发胶质瘤患者可选的治疗药物。

 Objective  To investigate the effect of methotrexate (MTX) on the localization of RAS protein and the downstream signal pathway and to explore the mechanism of MTX to inhibit the growth of human glioblastoma cell line U87. Methods  After the treatment of U87 cells with different concentrations of MTX (75nmol/L, 375nmol/L, 750nmol/L, 7,500nmol/L, 15,000nmol/ L), the cell viability and apoptosis rate were detected by MTT and FCM respectively. The distribution of RAS protein and activation was observed by confocal microscopy and the expression level of the MAPK/ERK protein in the downstream was measured by immune blotting method after U87 cell transduced retrovirus carrying RAS-GFP fusion gene. Further, the effect of MTX on the level of phosphorylation of P-p42/p44 MARK protein and the level of its regulation of the transcription factor c-MYC expression using different MAKP/ERK signaling pathway specific inhibitors (U0126 and PD98059) was investigated. The expression level of CD47 on the surface of cells was further assayed by FCM on U87 cells treated with low dose of MTX at different time points. Results  The different concentrations of MTX (75nmol/L, 375nmol/L, 750nmol/L, 7,500nmol/L, 15,000nmol/L) could inhibit the proliferation of human glioblastoma cell line U87 in a dose-dependent manner but can not directly induce apoptosis. The cell cycle of most U87 treated with the different concentrations of MTX was arrested at G2/S compared to untreated U87 cells. After U87 cells transduced RAS-GFP fusion gene (100% GFP) were treated with U87compared with the control group, the RAS protein was mislocation from the cell membrane to the cytoplasm; the P-p42/p44 MARK level was inhibited (P<0.05); the expression level of the transcription factor c-MYC was down-regulated (P<0.05). More important we addressed that CD47 related to glioblastoma growth and metastasis on the cell surface of U87 treated with MTX was decreased at different times, which confirmed by flow cytometry. Conclusions  It is the first time that we have demonstrated that MTX can inhibit the growth of glioblastoma cell line the U87; cell cycle was arrested at G2/S; the localization of the RAS protein was changed; RAS/MAPR/ERK/C-MYC signaling pathway was inhibited; MTX may be an alternative therapeutic drug for patients with recurrent glioblastoma.