华支睾吸虫重组14-3-3ε蛋白对胆管癌CCLP-1细胞迁移侵袭的作用研究

Effect of Clonorchis sinensis 14-3-3 epsilon recombinant protein on the migration and invasion of cholangiocarcinoma CCLP-1 cells

目的探讨华支睾吸虫重组14-3-3ε蛋白(r Cs14-3-3ε)对肝内胆管癌细胞株CCLP-1细胞波形蛋白(vimentin)表达的影响,观察该过程中CCLP-1细胞侵袭和迁移能力的变化。方法用不同浓度(1、5、10μg/ml)r Cs14-3-3ε与CCLP-1细胞共孵育24、48、72 h后,通过Q-PCR、Western blot等实验方法检测vimentin的表达水平。用划痕愈合实验和Transwell小室侵袭实验检测r Cs14-3-3ε对CCLP-1细胞迁移和侵袭能力的影响。结果 Q-PCR实验表明5μg/ml r Cs14-3-3ε刺激CCLP-1细胞72 h后及10μg/ml r Cs14-3-3ε刺激CCLP-1细胞24、48、72 h后,vimentin m RNA水平显著上调;Western blot实验显示,10μg/ml r Cs14-3-3ε刺激CCLP1细胞48、72 h后,vimentin蛋白水平显著上调。划痕实验表明,r Cs14-3-3ε(10μg/ml)能够促进细胞的迁移能力,处理后的细胞迁移率增大(P<0.01);Transwell小室检测显示,r Cs14-3-3ε(10μg/ml)处理组中穿膜细胞数为对照组的1.78倍,显著增加(P<0.05)。结论 r Cs14-3-3ε可以通过上调vimentin蛋白的表达,从而促进肝内胆管癌细胞株CCLP-1的侵袭和迁移能力。

Objective To explore the vimentin expression of cholangiocarcinoma CCLP-1 cells treated with recombinant Clonorchis sinensis 14-3-3ε protein（r Cs14-3-3ε）,investigate the effects of r Cs14-3-3ε on the migration and invasion abilities of CCLP-1 cells,and explore the possible role of vimentin in the migration and invasion mechanisms. Methods CCLP-1 cells were treated with different concentrations（1, 5, 10 μg / ml） of r Cs14-3-3ε at three time points. The cells without or with Cs ESPs were harvested at 24, 48, and 72 h. The expressions of vimentin were detected by Q-PCR and Western blot. The migration and invasion abilities of CCLP-1 cells were determined by wound healing assay and Transwell assay, respectively. Resu lts In Q-PCR analysis, the m RNA level of vimentin significantly increased in CCLP-1 cells treated with 5 μg / ml r Cs14-3-3ε at 72 hours. There was a prominent increase of vimentin m RNA level in 10 μg / ml of r Cs14-3-3ε-treated cells compared with those of the control at 24 h, 48 h and 72 h.Western blotting showed that the protein levels of vimentin significantly increased in CCLP-1 cells treated with 10 μg / ml r Cs14-3-3ε compared with those of the control at48 and 72 h. Wound healing assay showed that the migration rate of CCLP-1 cells which treated with 10 μg / ml r Cs14-3-3εsignificantly increased（P〈0.01）. The invasion assay showed that the numbers of CCLP-1 cells treated with 10 μg / ml r Cs14-3-3ε invading through the chamber was1.78-folds of the control cells（P〈0.05）. Conclusion r Cs14-3-3ε can enhance the invasion and migration abilities of CCLP-1 cells and may be through up-regulation of vimentin.