稳定表达人类疱疹病毒6型U94基因血管内皮细胞株的建立及U94对内皮细胞增殖和血管生成能力的影响

Construction of vascular endothelial cell line stably expressing HHV-6 U94 gene and the effect of U94 on endothelial cell proliferation and angiogenesis ability

目的:构建人类疱疹病毒6型U94基因慢病毒载体,研究U94基因对血管内皮细胞增殖及血管生成的影响。方法:以质粒pSR2PH-U94为模板,PCR扩增U94-6×His片段,克隆至慢病毒载体pLenti6.3-MCS-IRES2-EGFP,经酶切和测序鉴定后,将重组质粒与慢病毒辅助包装元件质粒共转染293T细胞,获得含U94-6×His基因的重组慢病毒。重组慢病毒感染血管内皮细胞EA.hy926,经Blasticidin筛选,RT-PCR和Western blot鉴定,获得稳定表达细胞株。CCK-8和小管形成实验研究U94基因对血管内皮细胞的增殖及血管生成能力的影响。结果:成功构建含U94基因的慢病毒表达载体pLenti-U94-IRES2-EGFP,重组慢病毒经包装、纯化后测得滴度为2.35×107TU/ml。重组慢病毒感染血管内皮细胞EA.hy926,获得能稳定表达U94基因的细胞株EA.hy926-U94。CCK-8及小管形成实验结果显示EA.hy926-U94细胞与阴性对照细胞EA.hy926-NC、正常EA.hy926细胞相比,细胞增殖活性明显降低,小管形成能力变差。结论:成功建立了慢病毒介导的稳定表达U94基因的血管内皮细胞株,研究表明U94可以抑制内皮细胞的增殖及血管生成。

Objective：To construct a lentiviral vector containing HHV-6 U94 gene and observe the effect of U94 on proliferation and angiogenesis of vascular endothelial cells.Methods：The U94-6×His fragment was amplified from plasmid pSR2PH-U94 by PCR and cloned into the lentiviral vector（pLenti6.3-MCS-IRES2-EGFP）.The recombinant plasmid was identified by restriction enzyme digestion and gene sequencing,and then cotransfected 293T cells with the auxiliary packaging components plasmids to obtain recombinant lentivirus containing U94-6×His gene.Endothelial cells EA.hy926 were infected with the recombinant lentivirus.After the Blasticidin screening and identification by RT-PCR and western blot,the resistant cell clones were selected.The proliferation and tube formation capacity of endothelial cells were examined by CCK-8 and tube formation assay respectively.Results：The U94 gene was successfully cloned to lentiviral vector pLenti-U94-IRES2-EGFP.The recombinant vector was packaged into 293T cells and the titer of the purified recombinant lentivirus was 2.35 × 107 TU/ml.The U94 stably expressing cell line EA.hy926-U94 was obtained after transfection with the recombinant lentivirus and Blasticidin screening.CCK-8 and tubule formation experimental results show that the proliferation and angiogenesis ability of EA.hy926-U94 were deteriorated significantly compared to the negative control cells EA.hy926-NC and normal cells EA.hy926.Conclusion：The pLenti-U94-IRES2-EGFP lentivitral expression vector was constructed and stably expressing human herpersvirus 6 U94 gene EA.hy926-U94 cell line was established successfully.U94 could inhibit endothelial cells proliferation and angiogenesis.