蟾蜍灵抑制食管癌细胞增殖、迁移和趋化运动的机制研究  

Influence of bufalin in proliferation, migration and chemotaxis of esophageal carcinoma cells

作　　者：田志辉[1] 张培[1] 曹彦坤[1] 刘月平[1] 

机构地区：[1]河北医科大学第四医院,河北石家庄050011

出　　处：《中国现代医学杂志》2015年第20期45-49,共5页China Journal of Modern Medicine

摘　　要：目的通过培养人食管癌TE13细胞株,观察蟾蜍灵对食管鳞状细胞癌细胞增殖、迁移、趋化运动及Raf-1、磷酸化Raf-1(p-Raf-1)表达的影响,阐明蟾蜍灵抑制食管癌转移的作用机制,为临床治疗提供实验依据。方法培养TE13细胞,通过MTT方法检测细胞药物毒性及对细胞增殖的影响,通过细胞划痕试验检测蟾蜍灵对迁移的抑制作用。通过Boyden小室实验检测蟾蜍灵处理后的食管癌细胞株TE13的趋化运动。采用免疫组织化学和Western blot检测蟾蜍灵处理后Raf-1、p-Raf-1蛋白的表达。结果 MTT结果显示,TE13细胞的增值抑制率随着蟾蜍灵浓度的增加(10、25、50、100和200 nmol/L)而升高(P<0.05)。划痕实验结果显示,不同溶度的蟾蜍灵处理TE13细胞后,TE13细胞的迁移能力随着蟾蜍灵浓度的增加而降低。Boyden小室实验结果显示,10、25、50和100 nmol/L蟾蜍灵处理后的食管癌细胞穿过滤膜细胞数量与对照组比较明显减少(P<0.05)。Western blot结果显示,各组Raf-1蛋白的表达比较差异无统计学意义(P>0.05)。25、50和100 nmol/L蟾蜍组p-Raf-1的表达量与对照组比较明显减少(P<0.05)。免疫组织化学检测结果显示,各组Raf-1蛋白的表达差异无统计学意义(P>0.05)。25、50和100 nmol/L蟾蜍灵组的p-Raf-1阳性细胞数及棕黄色颗粒数与对照组比较明显减少(P<0.05)。结论当蟾蜍灵的药物浓度<200 nmol/L时,食管癌TE13细胞增殖的抑制作用不能通过其细胞毒性实现。蟾蜍灵对食管鳞状细胞癌TE13细胞株的迁移、趋化运动能力有明显的抑制作用。蟾蜍灵不抑制Raf-1蛋白表达,但可以降低p-Raf-1在食管鳞状细胞癌TE13细胞株的表达,提示可能通过抑制Raf-1的激活抑制ERK通路,从而抑制食管鳞状细胞癌TE13细胞株的转移。[Objective] To investigate the effect of bufalin on proliferation, migration, chemotaxis and expressions of Raf-1 and p-Raf-1 in human esophageal squamous carcinoma cells through culture of the human esophageal cancer cell line TEl3, to clarify the inhibition of esophageal cancer metastasis by bufalin and to offer reasonable experimental evidence in the clinic therapy. [Methods] TEl3 cells were cultured. The cytotoxic effect of the drug on the ceils and the proliferation inhibition rate were detected by MTT method. Wound healing assay was performed to find the effect of bufalin on cell migration. The chemotaxis of TE13 ceils with bufalin was investigated by Boyden cabin. Western blot and immunocytochemistry were performed to detect the protein expressions of Raf-1 and p-Raf-1 in TEl3 cells with bufalin. [Results] MTT method showed that when different concentrations of bufalin （10, 25, 50, 100 and 200 nmol/L） were used to treat TEl3 cell hnes, the proliferation inhibition rate increased to various degrees, the differences were statistically significant （P 〈 0.05）. Wound healing assay showed that the inhibition of cell migration increased with the increasing bufalin concentrations after 24 h, Boyden cabin showed that the migration number of the cells through the transwell significantly reduced with the increasing bufalin concentrations （10, 25, 50 and 100 nmol/L） （P〈 0.05）. Western blot results showed no significant differences in Raf-1 protein expression among different groups （P〉 0.05）. The expression of p-Raf-1 of TE13 cells in 25, 50 and 100 nmol/L bufalin was decreased compared with the control group （P〈 0.05）. The immunocytochemistry results showed protein expression of Raf-1 in different groups had no significant differences （P〉0.05）. The positive cells and brown yellow particles of p-Raf-1 in the 25, 50 and 100 nmol/L bufalin groups were obviously decreased compared with those in the control group （P 〈 0.05）. [Conclusions] When bufalin concentration is below 200 n

关 键 词：食管鳞状细胞癌 RAF-1 磷酸化Raf-1 TE13细胞株 蟾蜍灵 细胞迁移 

分 类 号：R365[医药卫生—病理学] R735.1[医药卫生—基础医学]