hSulf-1基因腺病毒表达载体的构建及其对血管内皮细胞增殖、迁移能力的影响

Construction of adenovirus vector carrying hSulf-1 gene and its effects on proliferation and migration of human endothelial cells

目的构建携带人硫酸酯酶1基因(hSulf-1)的腺病毒载体,研究其对于人脐静脉内皮细胞ECV-304增殖、迁移能力的影响。方法通过基因操作技术将hSulf-1基因插入到腺病毒基因组中,构建重组腺病毒Ad5-hSulf1;应用蛋白质免疫印迹法检测hSulf-1蛋白在ECV-304细胞中的表达及细胞内磷酸化Akt、ERK的表达变化;MTT实验检测hSulf-1过表达对于ECV-304细胞增殖的影响;采用划痕实验研究hSulf-1过表达对于ECV-304细胞迁移的影响。结果成功构建携带目的基因hSulf-1的腺病毒载体;免疫印迹结果表明hSulf1基因过表达会下调ECV-304细胞Akt和ERK信号分子的磷酸化水平;细胞增殖实验结果表明hSulf1基因过表达抑制了ECV-304细胞增殖,感染复数为50、100时细胞存活率下降至(68.49±0.05)%以及(67.78±0.06)%(P<0.05);划痕实验结果表明hSulf1基因过表达能够抑制细胞的迁移,相比于对照组细胞迁移能力减弱(P<0.01)。结论重组腺病毒Ad5-hSulf1介导的hSulf-1基因在ECV-304细胞中的过表达明显抑制细胞增殖及迁移,为hSulf-1用于肿瘤及血管生长相关疾病的基因治疗奠定了基础。

Objective To construct a recombinant adenovirus vector carrying hSulf-1 gene and to investigate its effect on the proliferation and migration of human umbilical vein endothelial cells.Methods The hSulf-1 protein was inserted into adenovirus genome to generate recombinant adenovirus Ad5-hSulf1,and then the virus was used to infect ECV-304 cell line.The expression of hSulf-1 protein was detected by Western blotting analysis,the cell viability was examined by MTT assay,and the cell migration ability was evaluated by wound-healing assay in ECV-304 cells.Results The recombinant adenovirus Ad5-hSulf1 was successfully constructed.Western blotting analysis showed that over-expression of hSulf1 down-regulated the phosphorylation levels of Akt and ERK in ECV-304 cells.MTT assay showed that over-expression of hSulf1 inhibited the proliferation of ECV-304 cells,with the cell survival rate decreased to（68.49±0.05）% at MOI of 50 pfu/ml and（67.78±0.06）% at MOI of 100 pfu/ml（P0.05）.Wound-healing assay showed that over-expression of hSulf1 significantly inhibited the migration of ECV-304 cells compared with the control group（P0.01）.Conclusion The recombinant adenovirus Ad5-hSulf1-mediated hSulf-1 over-expression can markedly inhibit the proliferation and migration of ECV-304 cells,laying a foundation for hSulf-1 related gene therapy of tumor and angiogenesis-associated diseases.