溶瘤病毒H101联合照射对人肺腺癌细胞系A549凋亡及毒性的研究

Combination effect of oncolytic virus H101 and radiation on apoptosis induction and toxicity in A549 lung adenocarcinoma cells

目的 探讨溶瘤病毒（H101）与射线对人肺腺癌细胞系A549凋亡及毒性的影响。方法 体外培养人肺癌细胞系A549，取对数生长期的肺癌细胞，分为对照组（PBS）、单纯照射组（IR组）、溶瘤病毒组（H101组）、照射联合溶瘤病毒组（IR＋H101组）。采用膜联蛋白异硫氰酸荧光素/碘化丙锭（Annexin V-FITC/PI）进行双染，流式细胞仪检测各组细胞凋亡情况；采用乳酸脱氢酶（LDH）释放实验检测各组细胞毒性；用实时荧光定量RT-PCR检测H101表面衣壳蛋白Hexon mRNA表达并比较病毒复制情况。结果 H101组细胞凋亡率（55.37%）与对照组细胞凋亡率（1.03%）比较，差异有统计学意义（t=36.51，P〈0.05）；IR＋H101组细胞凋亡率（93.06%）与H101组（55.37%）、IR组（12.67%）以及对照组（1.03%）比较，差异均有统计学（t=13.51、24.14、38.99，P〈0.05）；与对照组比较，IR组和H101组在各个时间段的细胞LDH释放百分比差异有统计学意义（t=25.84、39.38、32.51、78.18，P〈0.05；t=31.40、2.68、23.43、60.98，P〈0.05）；IR＋H101组细胞LDH释放百分比与对照组比较（t=80.71、119.74、109.80、123.94，P〈0.05）、IR组（t=28.80、54.34、72.34、61.91，P〈0.05）和H101组（t=42.02、57.45、57.01、58.83，P〈0.05），差异均有统计学意义；与H101组比较，IR ＋ H101组Hexon的mRNA表达量在24、48和72 h分别增长了16.26、28.37和39.58倍，差异有统计学意义（t=54.50、33.73、29.28，P〈0.05）。结论 H101对肿瘤细胞具有放射增敏作用，同时射线也增强了H101的溶瘤作用，两者联合协同互补对肿瘤细胞起到杀伤作用。

Objective To investigate the apoptosis and toxicity of oncolytic virus H101 combined with radiation on apoptosis of A549 lung adenocarcinoma cells.Methods A549 lung adenocarcinoma cells in exponential growth phase were divided into four groups： control （PBS） group, radiation （IR） group, oncolytic virus （H101） group and radiation combined with oncolytic virus （IR＋H101） group. The cells were double dyed with Annexin fluorescein isothiocyanate （V-FITC/PI） and then the apoptosis ratio of cells in every group was detected by the flow cytometry. The cytotoxic effect of cells in every group was detected by lactate dehydrogenase （LDH） release test. The mRNA expression of oncolytic viruses H101 capsid protein Hexon was detected by real-time fluorescence PCR （RT-PCR） to compare the oncolytic virus replication in each group.Results Cell apoptosis rate in H101 group （55.37%） was significantly higher than that in PBS group （1.03%） （t=36.51, P〈0.05）. Cell apoptosis rate in IR＋H101 group （93.06%） was significantly higher than that in H101 group （55.37%）, IR group （12.67%） and PBS group （1.03%） （t=13.51, 24.14, 38.99, P〈0.05）. LDH releasing percentage in IR group and H101 group at different time after virus transfection was significantly higher than that in PBS group （t=25.84, 39.38, 32.51, 78.18, P〈0.05;t=31.40, 2.68, 23.43, 60.98, P〈0.05）. LDH releasing percentage in IR＋H101 group was significantly higher than that in PBS group （t=80.71, 119.74, 109.80, 123.94, P〈0.05）, IR group （t=28.80, 54.34, 72.34, 61.91, P〈0.05） and H101 group （t=42.02, 57.45, 57.01, 58.83, P〈0.05）. Compared with H101 group at the same time point, the mRNA expression of Hexon in IR＋H101 group at 24, 48 and 72 h was increased by 16.26, 28.37 and 39.58 times, respectively （t=54.50, 33.73, 29.28, P〈0.05）.Conclusions The oncolytic virus H101 plays a role of radiosensitization in tumor cells. Radiation also increases the replication of the oncolytic virus H101 and thereby enhances the oncolytic effect of the oncolytic virus H101. Therefore, oncolytic virus H101 combined with radiotherapy has synergistic effect on killing tumor cells.