华支睾吸虫severin蛋白对肝癌细胞SMMC-7402增殖、迁移及侵袭作用的初步研究

Effect of Clonorchis sinensis severin protein on the proliferation,migration and invasion of hepatocarcinoma SMMC-7402 cells

目的研究华支睾吸虫Severin蛋白对肝癌细胞株SMMC-7402增殖以及侵袭迁移能力的影响。方法构建Csseverin的慢病毒表达载体,并稳定转染肝癌SMMC-7402细胞。Western blot鉴定得到稳定过表达Csseverin的肝癌细胞系(PEZ-LV203-Csseverin 7402),并通过荧光显微镜观察其细胞内绿色荧光的表达;CCK-8实验检测过表达Csseverin后肝癌细胞SMMC-7402增殖能力的改变;用划痕愈合实验和Transwell小室侵袭实验检测Csseverin对SMMC-7402细胞迁移侵袭能力的影响;RT-PCR检测PEZ-LV203-Csseverin 7402和对照细胞内侵袭相关指标基质金属蛋白酶2(MMP-2)、MMP-9的m RNA水平。结果肝癌细胞SMMC-7402稳定过表达Csseverin后,Western blot检测出PEZ-LV203-Csseverin 7402中的Csseverin蛋白明显增多,荧光显微镜观察细胞有明显的绿色荧光表达;CCK-8实验表明Csseverin能明显促进SMMC-7402细胞的增殖能力。细胞划痕实验显示与对照细胞相比,过表达Csseverin的SMMC-7402细胞的迁移能力明显增强。Transwell小室检测显示,过表达Csseverin后的SMMC-7402中穿膜细胞数高于对照细胞组。RT-PCR检测出PEZ-LV203-Csseverin-7402中的MMP-2和MMP-9 m RNA水平明显高于对照细胞组。结论 Csseverin可促进肝癌细胞SMMC-7402的增殖,可能通过上调MMP-2和MMP-9表达从而促进肝癌细胞SMMC-7402的侵袭迁移能力。

Objective To explore the effects of Clonorchis sinensis severin protein（Csseverin） on the proliferation,migration and invasion abilities of hepatocarcinoma SMMC-7402 cells. Methods Csseverin expression plasmid was constructed and used to established the Csseverin-overexpressing SMMC-7402 Cells（PEZ-LV203-Cs sevein 7402）. The PEZ-LV203-Csseverin 7402 Cells were identified of the Csseverin by western blotting and fluorescent microscope. The proliferation of PEZ-LV203-Csseverin 7402 was quantified by CCK-8 assay. The migration and invasion abilities of PEZLV203-Csseverin 7402 were determined by wound healing assay and transwell assays respectively. The m RNA levels of matrix metalloproteinase 2（MMP-2） and MMP-9 were detected by RT-PCR. Results Csseverin protein was detected in the Csseverin-overexpressing SMMC-7402 cells（PEZ-LV203-Cssevein 7402）.The recombinant Csseverin can significantly promote the proliferation of SMMC-7402 cells, the migration and invasion abilities of PEZ-LV203-Cs severin 7402.m RNA levels of MMP-2 and MMP-9 were significantly increased in PEZ-LV203-Csseverin 7402 cells. Conclusion Csseverin could enhance the proliferation of SMMC-7402, and promote the invasion and migration abilities of SMMC-7402 cells by increasing expression of MMP-2 and MMP-9.