目的：探讨蟾蜍灵通过抑制人食管鳞癌细胞Raf／MEK／细胞外信号调节激酶（ ERK）信号通路的活化从而影响人食管鳞癌细胞增殖及迁移的作用机制。方法培养人食管鳞癌细胞株TE13，分为对照组、不同浓度蟾蜍灵组（蟾蜍灵10 nmol／L组、蟾蜍灵25 nmol／L组、蟾蜍灵50 nmol／L组、蟾蜍灵100 nmol／L组）、 UO126组和蟾蜍灵100 nmol／L＋UO126组，采用Western blotting方法和免疫细胞化学方法分别检测各组Raf-1、磷酸化Raf-1（ p-Raf-1）、MEK、磷酸化MEK （p-MEK）、 ERK、磷酸化细胞外信号调节激酶（p-ERK）表达量和阳性表达率。划痕实验和Boyden小室实验检测各组细胞的迁移距离和穿过滤膜的细胞数量。结果蟾蜍灵25 nmol／L组、蟾蜍灵50 nmol／L组和蟾蜍灵100 nmol／L组p-Raf-1、 p-ERK表达量低于对照组（ P＜0.05）；蟾蜍灵50 nmol／L组和蟾蜍灵100 nmol／L组p-MEK表达量低于对照组（ P ＜0.05）；蟾蜍灵100 nmol／L ＋UO126组p-Raf-1、 p-MEK、 p-ERK表达量低于UO126组（ P＜0.05）。蟾蜍灵25 nmol／L组、蟾蜍灵50 nmol／L组和蟾蜍灵100 nmol／L组p-Raf-1、 p-ERK阳性表达率低于对照组（P＜0.05）；蟾蜍灵50 nmol／L组和蟾蜍灵100 nmol／L组p-MEK阳性表达率低于对照组（P＜0.05）；蟾蜍灵100 nmol／L＋UO126组p-Raf-1、 p-MEK、 p-ERK阳性表达率低于UO126组（ P＜0.05）。划痕实验结果显示，蟾蜍灵25 nmol／L组、蟾蜍灵50 nmol／L组和蟾蜍灵100 nmol／L组细胞迁移距离短于对照组（ P＜0.05）；蟾蜍灵100 nmol／L＋UO126组细胞迁移距离短于UO126组（P＜0.05）。 Boyden小室实验结果显示，蟾蜍灵25 nmol／L组、蟾蜍灵50 nmol／L组和蟾蜍灵100 nmol／L组穿过滤膜的细胞数量少于对照组（ P＜0.05）；蟾蜍灵100 nmol／L＋UO126组穿过滤膜的细胞数量少于UO126组（ P＜0.05）。结论蟾蜍灵可下调细胞内p-Raf-1、 p-MEK、 p-ERK表达量，提示蟾蜍灵可能是通过抑制Raf／MEK／ERK信号通路活化这一过程而发挥作用，从而抑制人食管鳞癌细胞的生长。蟾蜍灵对人食管鳞癌细胞株TE13的迁移和趋化运动能力具有明显的抑制作用。%Objective To explore the mechanism of Bufalin affecting the proliferation and migration of human esophageal carcinoma cells through inhibiting the activity of Raf/MEK/ERK pathway.Methods The esophageal carcinoma cell lines TE13 were divided into control group, bufalin 10 nmol/L group, bufalin 25 nmol/L group, bufalin 50 nmol/L group, bufalin 100 nmol/L group, Uo126 group and bufalin 100 nmol/L +UO126 group, Western blotting and immunocytochemistry were performed to detect the expression quantities of Raf-1/p-Raf-1, MEK/p-MEK and ERK/p-ERK in different groups of TE13 cells, and positive expression rates of Raf-1/p-Raf-1, MEK/p-MEK and ERK/p-ERK were calculated.Wound Healing assay and Boyden cabin experiment were used to explore the cell migration distance in different cell groups and the number of cells which had penetrated filter membrane.Results The expression quantities of p-Raf-1 and p-ERK in bufalin 25 nmol/L group, bufalin 50 nmol/L group and bufalin 100 nmol/L group were significantly fewer than those in control group, respectively (P<0.05) .The expression quantities of p-MEK in bufalin 50 nmol/L group and bufalin 100 nmol/L group were significantly fewer than those in control group, respectively (P<0.05) .The expression quantities of p-Raf-1, p-MEK and p-ERK in bufalin 100 nmol/L+UO126 group were significantly fewer than those in UO126 group, respectively ( P<0.05 ) . The positive expression rates of p-Raf-1 and p-ERK in bufalin 25 nmol/L group, bufalin 50 nmol/L group and bufalin 100 nmol/L group were significantly lower than those in control group, respectively ( P<0.05 ) .The positive expression rates of p-MEK in bufalin 50 nmol/L group and bufalin 100 nmol/L group were significantly lower than those in control group, respectively (P<0.05) .The positive expression rates of p-Raf-1, p-MEK and p-ERK in bufalin 100 nmol/L+UO126 group were significantly lower than those in control group, respectively ( P <0.05 ) .According to results of Wound Healing assay, the cell migration distance in bufalin 25 nmol/L group, bufalin 50 nmol/L group and bufalin 100 nmol/L group was significantly shorter than that in control group, respectively ( P <0.05 ); the cell migration distance in bufalin 100 nmol/L+UO126 group was significantly shorter than that in UO126 group ( P <0.05 ) .According to results of Boyden cabin experiment, the number of cells which had penetrated filter membrane in bufalin 25 nmol/L group, bufalin 50 nmol/L group and bufalin 100 nmol/L group was significantly fewer than that in control group ( P <0.05 ); the number of cells which had penetrated filter membrane in bufalin 100 nmol/L+UO126 group was significantly fewer than that in UO126 group (P<0.05). Conclusion Bufalin can reduce the expression quantities of p-Raf-1, p-MEK and p-ERK.Bufalin may inhibit esophageal carcinoma cells growth though inhibiting the activity of Raf/MEK/ERK pathway.Bufalin can inhibit the migration and chemotaxis of human esophageal carcinoma cell lines TE13.
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