转化生长因子-β1与其受体阻断剂作用于人胆管癌细胞株后对转化生长因子-Smad轴的影响

Effect of transforming growth factor-β1 and transforming growth factor-β receptor blocker on the transforming growth factor-Smad axis in RBE cells

目的研究转化生长因子-β1（TGF-β1）相关信号通路在胆管癌细胞增殖、迁移和侵袭中的作用。方法对胆管癌细胞株RBE细胞单独或联合应用TGF-β1及其受体阻断剂（TGFβRI）SB525334/SB431542，根据单独或联合用药共分为6个组。（1）观察细胞形态学改变。（2）Trasswell法检测RBE细胞迁移及侵袭性变化。（3）Western blot检测Smad4、E-钙黏蛋白（E-cadherin）、N-钙黏蛋白（N-cadherin）及Slug的蛋白表达变化。计量资料采用均数±标准差（±s）表示，多组间比较采用单因素方差分析（One-way ANOVA）。结果（1）TGF-β1引发RBE细胞的形态由上皮样向间质样转变。（2）Transwell法迁移性改变：空白对照组为（85.66±7.76）个，TGF-β1组为（118.17±12.69）个，SB431542组为（53.00±5.51）个、TGF-β1＋SB431542组为（86.33±9.67）个，SB525334组为（52.50±7.45）个，TGF-β1＋SB525334组为（81.00±7.62）个。TGF-β1组比空白对照组，迁移细胞数目明显增多（P＝0.000），SB525334组、SB431542组比空白对照组，迁移细胞数目明显减少（P值均为0.000）。Transwell法检测侵袭性改变：空白对照组为（73.83±9.47）个，TGF-β1组为（127.83±7.33）个，SB431542组为（59.83±7.28）个、TGF-β1＋SB431542组为（79.67±7.31）个，SB525334为（55.67±7.26）个，TGF-β1＋SB525334组为（76.00±8.51）个。TGF-β1组比空白对照组，侵袭细胞数目明显增多（P＝0.000），SB525334组、SB431542组比空白对照组，侵袭细胞数目明显减少（P值分别为0.000、0.003）。（3）Western blot实验中TGF-β1组中Smad4蛋白的表达水平较空白对照组明显增高，差异有统计学意义（P＝0.000）。E-cadherin、N-cadherin、Slug蛋白的表达水平在TGF-β1组与空白对照组中比较差异均有统计学意义（其P值分别为0.044、0.001、0.001）；上述指标在SB431542组与空白对照组中比较差异均有统计学意义（其P值分别为0.001、0.000、0.000），其在SB525334组与空白对照组中比较差异均有统计学意义（其P值分别为0.001、0.000、0.000）。结论（1）TGF-β1可诱导RBE细胞的形态由上皮样向间质样转变。（2）TGF-β1可以促进REB细胞的迁移和侵袭作用，而TGFβRI可以阻断REB细胞的迁移和侵袭作用。（3）TGF-β1及TGFβRI能够影响N-cadherin、E-cadherin及Slug蛋白表达的变化：TGF-β1可促进N-cadherin、Slug蛋白表达的增加，促进E-cadherin蛋白表达的降低；TGFβRI可降低N-cadherin、Slug蛋白的表达，促进E-cadherin蛋白表达。

Objective To study the effect of transforming growth factor-β1 （TGF-β1） related signaling pathway on the proliferation, migration and invasion of cholangiocarcinoma cells.Methods Cholangiocarcinoma RBE cells were treated with TGF-β1 and TGFβ receptor blocker SB525334/SB431542 alone or in combination. Six groups were set up. The morphological changes of cells were observed. Transwell assay was conducted to measure the effect of TGF-β1 and SB525334/SB431542 on the migration and invasion of RBE cells. Western blotting was applied to detect the expression of Smad4, E-cadherin, N-cadherin and Slug in RBE cells after treatment with TGF-β1 and SB525334/SB431542 alone or in combination. Measurement data were expressed as mean ± standard deviation （±s）, multiple groups were compared using one-way ANOVA.Results （1） TGF-β1 induced the epithelial-like to the interstitial transformation of RBE cells. （2） The number of migrating cells was 85.66±7.76 in the blank control group, 118.17±12.69 in the TGF-β1 group, 53.00±5.51 in the SB431542 group, 86.33±9.67 in the TGF-β1＋ SB431542 group, 52.50±7.45 in the SB525334 group, and 81.00±7.62 in the TGF-β1＋ SB525334 group. The number of migrating cells in the TGF-β1 group was significantly increased as compared with the blank control group （P=0.000）. The number of migrating cells in the SB525334 group and SB431542 group was significantly decreased as compared with the blank control group （P=0.000）. The number of invasive cells was 73.83±9.47 in the blank control group, 127.83±7.33 in the TGF-β1 group, 59.83±7.28 in the SB431542 group, 79.67±7.31 in the TGF-β1＋ SB431542 group, 55.67±7.26 in the SB525334 group, and 76.00±8.51 in the TGF-β1＋ SB525334 group. The numbers of cells in TGF-β1 group and blank control group were significantly increased （P=0.000）. The numbers of cells in SB525334 group and SB431542 group were significantly decreased compared with the blank control group （P values were 0.000, 0.003, respectively）. （3） Western blotting experiment. The expression level of Smad4 in TGF-β1 group was significantly higher than that in blank control group （P=0.000）. E-cadherin, N-cadherin, Slug protein levels were compared separately, the results showed that the different protein expression levels in TGF-β1 group and the control group were statistically significant differences （P values were 0.044, 0.001, 0.001, respectively）. There was significant difference between the SB431542 group and the blank control group （P values were 0.001, 0.000, 0.000, respectively）. There was significant difference between the SB525334 group and the blank control group （P values were 0.001, 0.000, 0.000, respectively）.Conclusion （1） TGF-β1 can induce the morphological changes of RBE cells from epithelial to interstitial transformation. （2） TGF-β1 can promote the migration and invasion of REB cells, while SB can block the migration and invasion of REB cells. TGF-β1 and TGFβRI could affect the expression of N-cadherin, E-cadherin and Slug protein. （3） TGF-β1 could promote the increase of N-cadherin and Slug protein expression and decrease the expression of E-cadherin protein. SB can reduce the expression of N-cadherin and Slug protein, and promote the expression of E-cadherin protein.