摘要
目的探讨Hint2基因过表达质粒的构建及其对肝癌细胞BEL-7402迁移的影响。方法根据Hint2基因设计引物,扩增Hint2基因全长并以pcDNA3.1(+)为载体构建Hint2过表达质粒。转染BEL-7402细胞提取m RNA和总蛋白,分别使用Real-time PCR和Western blot检测转染细胞Hint2 mRNA和蛋白质表达的变化,应用免疫荧光染色法对细胞中Hint2蛋白与细胞线粒体进行共定位。使用划痕愈合实验检测Hint2过表达后对肝癌细胞BEL-7402迁移能力的影响。结果转染Hint2过表达质粒细胞的Hint2 mRNA和蛋白表达显著上调,免疫荧光染色表明过表达的Hint2蛋白定位于线粒体。划痕愈合实验表明Hint2过表达质粒使肝癌细胞BEL-7402迁移速率显著降低。结论 Hint2过表达质粒能够上调BEL-7402细胞中Hint2m RNA和蛋白质的表达水平,表达的蛋白定位于细胞线粒体并抑制肝癌细胞的迁移。
Objective To investigate the construction of Hint2 overexpression plasmid and its effect on invasion capability of liver cancer cell BEL-7402. Methods According to the sequence of Hint2 gene, primers were designed to amplify Hint2 gene fragment for Hint2 gene overexpression plasmid construction. mRNA and total protein were extracted from Hint2 overexpressed BEL-7402 cells, real-time PCR and Western blot were applied to determine the expression levels of Hint2, and then the location of exogenous Hint2 in mitochondria was confirmed through immunorescence staining. Subsequently, wound healing experiment was taken to detect the changes of invasion and migration capability of BEL-7402 cells after Hint2 overexpression. Results The mRNA and protein expression levels of Hint2 up-regulated significantly in BEL-7402 cells after transfection with Hint2 overexpression plasmid. According to the result of immunofluorescence staining, the exogenous Hint2 protein was co-located with the mitochondria. Wound healing experiment showed the up-regulation of Hint2 restrained the migration ability in BEL-7402 cells. Conclusion After transfection with Hint2 overexpression plasmid, the expression level of Hint2, which was bound with mitochondria, was increased and the migration ability of BEL-7402 cells was restrained.
作者
李奇萌
林国南
LI Qi-meng;LIN Guo-nan(Capital Medical University,Beoing 100069,China)
出处
《中国肝脏病杂志（电子版）》
CAS
2018年第3期1-6,共6页
Chinese Journal of Liver Diseases：Electronic Version
基金
国家自然科学基金(31301204)
