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  

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

作　　者：李猛[1] 顾斌[1] 王金凤[2] 周锋[2] 李凌云[2] 李俊漾[1] 张国锋[1] 幸标[1] 张寒[1] 姚堃[2] 胡卫星[1] 

机构地区：[1]南京医科大学第一附属医院神经外科,江苏南京210029 [2]南京医科大学微生物与免疫学系,江苏南京210029

出　　处：《南京医科大学学报（自然科学版）》2013年第4期438-443,共6页Journal of Nanjing Medical University(Natural Sciences)

基　　金：国家自然科学基金资助(30972784;81273235;81201520)

摘　　要：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。Objective：To investigate the influences of human herpesvims 6 variant A DR7 gene on the proliferation, migration and invasion of human glioma U87 cells, nethods：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 x His-IRES2-EGFP lentivitral expression vector was constructed and UgT-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 significant- ly 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）. Conclnsion：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.

关 键 词：人类疱疹病毒6型 神经胶质瘤 DR7 增殖 迁移 侵袭 

分 类 号：R739.41[医药卫生—肿瘤]