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| 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 |
| 季卫丹1,包龙龙1,严妍2,曹璐2,傅晓辉2,姜小清2,苏长青1,2* |
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(1. 苏州大学医学部遗传学系,苏州 215123
2. 第二军医大学东方肝胆外科医院分子肿瘤研究室,上海 200438
*通信作者) |
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| 摘要: |
| 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。 |
| 关键词: 腺病毒 hSulf-1基因 血管 内皮细胞 细胞增殖 细胞运动 |
| DOI：10.3724/SP.J.1008.2012.00247 |
| 投稿时间：2011-12-14修订日期：2012-02-25 |
| 基金项目:国家科技重大专项(2009ZX09102-235),国家自然科学基金(81172019,81172303). |
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| Construction of adenovirus vector carrying hSulf-1 gene and its effects on proliferation and migration of human endothelial cells |
| JI Wei-dan1,BAO Long-long1,YAN Yan2,CAO Lu2,FU Xiao-hui2,JIANG Xiao-qing2,SU Chang-qing1,2* |
(1. Department of Medical Genetics, Medical College of Soochow University, Suzhou 215123, Jiangsu, China
2. Department of Molecular Oncology, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai 200438, China
*Corresponding author.) |
| Abstract: |
| 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(P<0.05). Wound-healing assay showed that over-expression of hSulf1 significantly inhibited the migration of ECV-304 cells compared with the control group (P<0.01). 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. |
| Key words: adenovirus hSulf-1 gene blood vessels endothelial cells cell proliferation cell movement |
