甲氨喋呤对血管平滑肌细胞增殖、迁移和凋亡的影响

The effects of methotrexate on vascular smooth muscle cells proliferation, migration and apoptosis

目的 观察甲氨喋呤 (MTX)对体外培养的血管平滑肌细胞增殖、迁移和凋亡的影响。方法 体外培养兔胸主动脉血管平滑肌细胞 ,在培养基中加入不同浓度的MTX ,采用细胞计数及检测细胞周期的方法观察MTX对细胞增殖的影响 ,采用细胞刮片的方法观察MTX对细胞迁移的作用 ,采用检测DNA片断化的方法观察细胞凋亡现象。结果 ①MTX在 2 5～ 10 0nmol·L-1的浓度范围内呈剂量依赖性地抑制VSMC增殖。② 2 5nmol·L-1及 5 0nmol·L-1MTX显著增加细胞周期中S期细胞的百分含量 ,减低G2 /M期细胞的百分含量 (P <0 0 5 ) ,10 0nmol·L-1MTX显著增加了G0 /G1期细胞的百分含量 (P <0 0 1)。③加入MTX的VSMC对血清刺激的迁移受到抑制。④ 10 0nmol·L-1及 2 0 0nmol·L-1的MTX作用于VSMC后 ,流式细胞仪检测亚二倍体的细胞数高于对照组 (P <0 0 1) ;DNA凝胶电泳可以见到梯形条带 ;TUNEL染色阳性细胞的百分数高于对照组 ,具有统计学差异 (P <0 0 1)。结论 甲氨喋呤能够抑制体外培养的血管平滑肌细胞增殖和迁移 ,并促进细胞凋亡。

Aim To observe the effect of methotrexate (MTX) on proliferation, migration and apoptosis of cultured vascular smooth muscle cells (VSMC). Methods Rabbit thoracaortic VSMC were cultured in vitro.VSMC proliferation was evaluated by cell counting and cell cycle analysis. Monolayer cell scrape was used to observe VSMC migration. Apoptosis was observed with flow cytometry, DNA gel electrophoresis and TUNEL stain. Results MTX (25～100 nmol·L -1) inhibited VSMC proliferation in a dose-dependent manner.25 nmol·L -1 and 50 nmol·L -1 MTX increased the percentage of the S phase cells and decreased the percentage of the G 2/M phase cells (P<0.05 vscontrol),while 100 nmol·L -1 MTX increased the percentage of the G 0/G 1phase cells in the cell cycle (P<0.01vscontrol). The migration distance of VSMC treated with MTX (25～100 nmol·L -1) was inhibited markedly.It was demonstrated by flow cytometry that the percentage of cells treated with MTX of sub-G 1 region was significantly increased. DNA ladder was seen after 72 hours treatment with 100 nmol·L -1 and 200 nmol·L -1 MTX. After incubated with MTX for 72 h,the cells stained by TUNEL had a significantly higher percentage than control (P<0.01).Conclusion MTX could not only suppress VSMC proliferation and migration, but also cause their apoptosis.