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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 the lentiviral vector of human herpesvirus type 6 U94 gene and study the effect of U94 gene on the proliferation and angiogenesis of vascular endothelial cells. METHODS: U94-6 × His fragment was amplified by PCR using plasmid pSR2PH-U94 as a template and cloned into the lentiviral vector pLenti6.3-MCS-IRES2-EGFP. After digestion and sequencing, the recombinant plasmid was co-transfected with lentivirus 293T cells were cotransfected with the packaging plasmid and the recombinant lentivirus containing U94-6xHis gene was obtained. The recombinant lentivirus infected the vascular endothelial cells EA.hy926 and identified by Blasticidin, and identified by RT-PCR and Western blot. Stably expressing cell lines were obtained. CCK-8 and Tubular Formation Experiments The effect of U94 gene on the proliferation and angiogenesis of vascular endothelial cells was investigated. Results: The lentiviral vector pLenti-U94-IRES2-EGFP containing U94 gene was constructed successfully. The recombinant lentivirus was packaged and purified and its titer was 2.35 × 107TU / ml. The recombinant lentivirus infected the vascular endothelial cells EA.hy926 and obtained the cell line EA.hy926-U94 stably expressing the U94 gene. The results of CCK-8 and tubule formation assay showed that compared with EA.hy926-NC and normal EA.hy926 cells of EA.hy926-U94 cells, the proliferation activity of EA.hy926-U94 cells was significantly decreased and the ability of tubular formation was worsened. CONCLUSION: The lentivirus-mediated endothelial cell line expressing U94 gene was established successfully. Studies have shown that U94 can inhibit endothelial cell proliferation and angiogenesis.