人类疱疹病毒6A亚型DR7基因对神经胶质瘤细胞U87增殖､迁移､侵袭能力的影响

目的:构建含人类疱疹病毒6A亚型DR7基因的慢病毒,研究DR7基因表达对神经胶质瘤细胞U87增殖?迁移?侵袭能力的影响?方法:PCR扩增DR7基因,克隆至慢病毒载体pLenti6.3-MCS-IRES2-EGFP,构建pLenti6.3-DR7-IRES2-EGFP重组慢病毒载体,经293T细胞包装重组病毒转染至神经胶质瘤细胞U87,经blasticidin筛选建立稳定表达株,通过细胞增殖实验?细胞周期实验?细胞划痕及Transwell实验研究稳定表达DR7基因对U87细胞的增殖?迁移及侵袭能力的影响?结果:成功构建了pLenti6.3-DR7-6 × His-IRES2-EGFP慢病毒表达载体,筛选了稳定表达DR7基因的U87-DR7-EGFP细胞株,CCK-8细胞增殖实验显示,稳定表达DR7基因的U87-DR7-EGFP细胞与阴性对照细胞U87-NC-EGFP?U87细胞相比,细胞增殖活性明显增高,差异具有统计学意义(P < 0.001)?细胞周期检测发现U87-DR7-EGFP细胞S?G2/M期细胞所占比例多于U87-NC-EGFP?U87细胞:S期的比例分别为(34.73 ± 1.12)%?(24.89 ± 0.93)%?(25.39 ± 0.96)%,差异具有统计学意义(P < 0.001);G2/M期分别为(17.35 ± 1.61)%?(11.36 ± 1.50)%?(13.17 ± 1.95)%,差异具有统计学意义(P < 0.05)?细胞划痕实验表明,U87-DR7-EGFP细胞愈合能力明显强于U87-NC-EGFP?U87细胞,划痕6 h愈合率分别为:(33.55 ± 2.83)%?(23.50 ± 3.18)%?(22.03 ± 1.47)%,差异具有统计学意义(P < 0.01);划痕12 h愈合率分别为(70.50 ± 5.39)%?(53.60 ± 4.67)%?(55.09 ± 2.83)%,差异具有统计学意义(P < 0.001)?Transwell实验表明,U87-DR7-EGFP细胞的穿膜数量明显多于U87-NC-EGFP?U87细胞,分别为:(543.00 ± 22.94)?(387.00 ± 15.63)?(412.00 ± 20.30)个,差异具有统计学意义(P < 0.001)?结论:人类疱疹病毒6A亚型DR7基因表达能在体外促进人神经胶质瘤细胞U87增殖?迁移及侵袭,提示其在神经胶质瘤的发生和发展中可能起一定作用?

Effect of human herpesvirus 6 variant A DR7 gene on cell proliferation, migration and invasion of human glioma U87 cells

Objective:To investigate the influences of human herpesvirus 6 variant A DR7 gene on the proliferation, migration and invasion of human glioma U87 cells. Methods:Lentiviral vectors pLenti6.3-DR7-IRES2-EGFP was constructed and then transfected to human glioma cells U87. A separate U87 cell line stably expressing DR7 was established after blasticidin screening. The cell proliferation was detected by cell counting Kit-8 and the cell cycle was mearsured by flow cytometry. The migration and invasion abilities were detected by wound healing assay and transwell assay, respectively. Results:The pLenti6.3-DR7-6 × His-IRES2-EGFP lentivitral expression vector was constructed and U87-DR7-EGFP cell line stably expressing DR7 gene was established successfully. Compared with U87-NC-EGFP and U87 cells, The cell proliferation of U87-DR7-EGFP cells was significantly increased. Flow cytometry showed that the overexpression of DR7 increased the proportion of U87 cells at S and G2/M phase compared with U87-NC-EGFP and U87 cells. The percentages of S phase were (34.73 ± 1.12)%, (24.89 ± 0.93)%, (25.39 ± 0.96)% (P < 0.001), and G2/M phase were (17.35 ± 1.61)%, (11.36 ± 1.50)%, (13.17 ± 1.95)% (P < 0.05), respectively. The cure rates were significantly increased in the U87-DR7-EGFP cells compared with U87-NC-EGFP and U87 cells: 6 h cure rates were(33.55 ± 2.83)%, (23.50 ± 3.18)%, (22.03 ± 1.47)% and 12 h cure rates were (70.50 ± 5.39)%, (53.60 ± 4.67)%, (55.09 ± 2.83)%, respectively. The average number of invasive cells in each visual field was significantly increased in the U87-DR7-EGFP cells compared with that of the U87-NC-EGFP and U87 cells (543.00 ± 22.94, 387.00 ± 15.63, 412.00 ± 20.30, P < 0.001). Conclusion:Human herpesvirus 6 variant A DR7 gene promote the proliferation, migration and invasion of human glioma cell U87 in vitro, suggesting that it may play a role in the development of glioma