派特灵对Ect1/E6E7细胞增殖迁移能力及Wnt/β-catenin通路的影响

Effects of Paiteling on proliferation and migration of Ect1/E6E7 cells and its effects on Wnt/β-catenin signaling pathway

目的:研究派特灵对Ect1/E6E7细胞增殖迁移能力及Wnt/β-catenin通路的影响。方法:以含10%胎牛血清的DMEM培养基培养细胞,将细胞分为对照组和派特灵组(3.906、2.604、1.953、1.563、1.302、1.116、0.977g/L),加药干预24h,镜下观察细胞形态变化,并采用MTT法检测细胞活力,计算派特灵对Ect1/E6E7细胞的半数抑制浓度(IC50);将Ect1/E6E7细胞分为对照组,顺铂组(0.01g/L),抑制剂组,派特灵高、中、低浓度组(2.974、1.487、0.991g/L),分别用CCK-8、划痕、Transwell方法检测派特灵对Ect1/E6E7细胞增殖迁移能力的影响;以及用免疫组化、Western Blotting方法检测各组细胞Wnt4、β-catenin蛋白的表达。结果:对照组细胞呈扁平多边形,顺铂组、派特灵各浓度组细胞皱缩、体积变小、数量减少,以派特灵高浓度组最明显;MTT实验显示,派特灵对Ect1/E6E7细胞增殖有明显抑制作用,IC50为2.974g/L;CCK-8法结果示顺铂组和派特灵高浓度组对Ect1/E6E7细胞增殖有较强抑制作用;划痕实验示派特灵高浓度组、顺铂组药物干预12h后,划痕面积均显著高于对照组(P<0.01,P<0.05),派特灵高浓度组划痕面积高于派特灵低浓度组(P<0.05);药物干预24h后,派特灵高浓度组、顺铂组划痕面积显著高于对照组和派特灵中、低浓度组(P<0.01);Transwell迁移实验示顺铂组、派特灵各浓度组细胞迁移数量显著低于对照组(P<0.01),顺铂组、派特灵高浓度组细胞迁移数量显著低于派特灵中、低浓度组(P<0.01);Western Blotting实验结果显示,与抑制剂XAV939组比较,对照组、顺铂组、派特灵各浓度组Wnt4蛋白表达量显著升高(P<0.01);派特灵高、中浓度组Wnt4蛋白表达量比派特灵低浓度组显著降低(P<0.01);Western Blotting、免疫组化实验结果显示与对照组比较,顺铂组、抑制剂组、派特灵各浓度组β-catenin蛋白表达量显著减少(P<0.01);与抑制剂XAV939组比较,顺铂组、派特灵各浓度组β-catenin蛋白表达量显著增加(P<0.01)。结论:派特灵可抑制Ect1/E6E7细胞增殖迁移能力且呈一定量效关系,可能通过抑制Wnt/β-catenin信号通路发挥作用。

Objective: To study the effect of Paiteling on the proliferation and migration of Ect1/E6 E7 cells and its effect on the Wnt/β-catenin pathway. Methods: Cells were cultured in DMEM medium containing 10% fetal bovine serum, and the cells were divided into blank group, Paiteling group(3.906, 2.604, 1.953, 1.563, 1.302, 1.116, 0.977 g/L), with drug intervention after 24 h, observing the changes of cell morphology under the microscope, and using the MTT method to detect cell viability, calculating the 50% inhibitory concentration(IC50) of Paiteling on Ect1/E6 E7 cells;divide Ect1/E6 E7 cells into control group and cisplatin group(0.01 g/L), inhibitor group, high, medium, and low concentration group of Paiteling(2.974, 1.487, 0.991 g/L), CCK-8, scratch, Transwell test method was used to detect Paiteling with the effect of Ect1/E6 E7 cells’ proliferation and migration ability;immunohistochemistry and Western blotting were used to detect the expression of Wnt4 and β-catenin protein in Ect1/E6 E7 cells. Results: The cells in the control group were flat polygons. The shrinkage volume and number of cells in the cisplatin group and the Paiteling group became smaller, and the number of cells in the Paiteling high concentration group was the most obvious. The MTT experiment showed that Paiteling had a significant inhibitory effect on Ect1/E6 E7 cells. The calculated IC50 of Paiteling on Ect1/E6 E7 cells is 2.974 g/L;the results of the CCK-8 method show that the cisplatin group and Paiteling high concentration group have a greater effect on the proliferation of Ect1/E6 E7 cells. The scratch test showed that, after drug intervention for 12 hours, the scratch areas of the high-concentration group of Paiteling and cisplatin group were significantly higher than that of the control group(P<0.01, P<0.05), and the area of scratches in the high-concentration group was higher than that of the control group. Trane low-concentration group(P<0.05);after 24 hours of drug intervention, the scratch area of Paiteling high-concentration group and cisplatin group were significantly higher than that of control group, Paiteling-medium and lowconcentration group(P<0.01). The Transwell migration experiment showed that the number of cell migration in each concentration group of cisplatin group and Paiteling was significantly lower than that of the control group(P<0.01), and the number of cell migration in the cisplatin group and Paiteling high concentration group was significantly lower than that of the middle and low concentrations of Paiteling group(P<0.01);Western Blotting results showed that, compared with the inhibitor XAV939 group, the Wnt4 protein expression in the control group, cisplatin group, and Paiteling increased significantly(P<0.01);The expression of Wnt4 protein in Paiteling high and medium concentration group were significantly lower than that in the low-concentration group(P<0.01);the results of Western Blotting and immunohistochemistry showed that compared with the control group, the cisplatin group, the inhibitor group, and every concentration in Paiteling group’s expression of β-catenin protein in the concentration group was significantly reduced(P<0.01);compared with the inhibitor XAV939 group, the expression of β-catenin protein in the cisplatin group and Paiteling each concentration group increased significantly(P<0.01). Conclusion: Paiteling can inhibit the proliferation and migration ability of Ect1/E6 E7 cells with a certain dose-effect relationship, which may play a role by inhibiting the Wnt/β-catenin signaling pathway.