腺病毒介导的白细胞介素24基因对人肺腺癌细胞的放疗增敏作用

Inhibitory and radiosensitizing effects of adenovirus-mediated IL-24gene on human lung adenocarcinoma

目的研究腺病毒介导的白细胞介素24基因(Ad-IL-24)对人肺腺癌细胞SPC-A1体外抑癌效应、放疗增敏作用。方法将Ad-IL-24感染的SPC-A1细胞,用逆转录-聚合酶链反应(RT-PCR)及蛋白印迹(Western blot)法检测IL-24目的基因在SPC-A1细胞中的转录和表达;四甲基偶氮唑蓝(MTT)法检测磷酸盐缓冲液(PBS)组、腺病毒(Ad-GFP)组、Ad-IL-24组、放疗组、Ad-GFP联合放疗组(Ad+放疗组)及Ad-IL-24联合放疗组(Ad-IL-24+放疗组)对SPC-A1细胞生长抑制作用,流式细胞术(FCM)检测各组SPC-A1细胞生长周期和凋亡率。RT-PCR法检测SPCA1细胞中生存素、半胱氨酸蛋白酶3(Caspase-3)、B细胞淋巴瘤/白血病基因伴随蛋白X(bax)、B细胞淋巴瘤/白血病基因-2(bcl-2)凋亡相关因子的表达。结果 Ad-IL-24作用SPC-A1细胞后,目的基因在SPC-A1细胞中成功转录及表达;Ad-IL-24能显著抑制SPC-A1细胞的生长且呈现时间依赖性,Ad-IL-24组(31.1±0.9)%、放疗组(44.4±2.3)%、Ad-IL-24+放疗组(72.4±1.5)%的生长抑制率分别与Ad-GFP组(2.7±1.1)%比较,均差异有统计学意义(P<0.01),且Ad-IL-24+放疗组优于Ad-IL-24组、放疗组(F=314.613,P<0.01),呈现放疗增敏的协同作用(Q=1.172);Ad-IL-24可诱导SPC-A1细胞G2/M期阻滞和细胞凋亡,Ad-IL-24+放疗组(40.6±3.3)%的细胞凋亡率与Ad-IL-24组(15.4±1.1)%、放疗组(26.3±1.7)%比较,差异有统计学意义(F=87.194,P<0.01),且具有放疗增敏协同作用(Q=1.162)。Ad-IL-24能明显上调促细胞凋亡相关因子bax、Caspase-3,并下调细胞抗凋亡因子bcl-2、生存素的表达,Ad-IL-24+放疗组作用优于Ad-IL-24组、放疗组。结论 Ad-IL-24有放疗增敏的作用,是理想的放疗增敏剂,该作用机制可能与诱导肿瘤细胞阻滞在G2/M期以及促进肿瘤细胞凋亡有关。

ABSTRACT：Objective To investigate the in vitro inhibitory and radiosensitizing effects of adenovirus-mediated interleukin-24（Ad-IL-24）gene on human lung adenocarcinoma cell SPC-A1.Methods SPC-A1 cells were divided into PBS group,Ad-GFP group,Ad-IL-24 group,radiotherapy group,Ad-GFP combined with radiotherapy group and Ad-IL-24 combined with radiotherapy group.Then,the expression of IL-24 in SPC-A1 cells were determined by reverse transcription-PCR（RT-PCR）and Western blot.The cell growth in each group was assessed by MTT assay and the cell cycle and apoptosis were determined by flow cytometry（FCM）.The expression of survivin,caspase-3,bax and bcl-2 were analyzed by RT-PCR.Results After Ad-IL-24 was successfully expressed in SPC-A1 cells,it could significantly inhibit the growth of SPC-A1 cells in a time-dependent manner.Compared with that in Ad-GFP group （2.7±1.1）%, the inhibitory rate increased significantly respectively in Ad-IL-24 group （31.1 ±0.9）%,radiotherapy group （44.4± 2.3）% and Ad-IL-24 combined with radiotherapy group （72.4± 1.5）% （P 〈0.01）.Especially,the inhibitory rate in Ad-IL-24 combined with radiotherapy group was more significant than that in the Ad-IL-24 group and the radiotherapy group （F =314.613,P 〈0.01;Q = 1.172）,showing prominently synergistic effects.Ad-IL-24 also could induce SPC-A1＆amp;nbsp;cells arresting at G2/M phase and eventually induced apoptosis.The apoptotic rate in Ad-IL-24 combined with radiotherapy group （40.6 ± 3.3 ）% was significantly higher than that in Ad-IL-24 group （15.4 ± 1.1 ）% and radiotherapy group （26.3±1.7）%（F =87.194,P 〈0.01）,showing notablely synergistic effect （Q = 1.162）.Ad-IL-24 could upregulate the expression of bax and caspase-3 and downregulate the expression of bcl-2 and survivin in SPC-A1 cells.Ad-IL-24 combined with radiotherapy group had more significantly effects than Ad-IL-24 group and radiotherapy group.Conclusion Ad-IL-24 can enhance the radiosensitizing effect in the antitumor treatment and may be an ideal radiosensitizing agent,and its underlying mechanisms may involve arresting tumor cells at the G2/M phase of cell cycle and,then inducing cell apoptosis.