辣根过氧化物酶/吲哚乙酸自杀基因系统联合放射对Hep-2细胞的生长抑制作用

Inhibitory Effect on Hep-2 Cells with Combination of Horseradish Peroxidase/Indole-3-Acetic Acid Suicide Gene Therapy System and Radiation

目的:研究辣根过氧化物酶(HRP)/吲哚乙酸(IAA)自杀基因系统联合放射对人喉鳞癌Hep-2细胞株的生长抑制作用,观察HRP/IAA系统是否具有放射增敏性。方法:将构建成功的pcDNA3.1-HRP转染Hep-2细胞,RT-PCR及Western blotting分别在mRNA和蛋白质两个水平检测其表达;分别以0.5 mmol/L IAA、2 Gyγ-射线及两者联合作用于转染HRP后的Hep-2细胞,实验分为4组:A、对照组;B、加药组;C、放射组;D、加药及放射组。克隆形成实验观察HRP/IAA系统对细胞存活分数的影响,通过拟合生存曲线计算放射增敏比SERSF2;流式细胞仪分析各组细胞周期时相的变化;AnnexinV-FITC试剂盒比较各组细胞凋亡率。结果:转染pcDNA3.1-HRP的Hep-2细胞,RT-PCR和Western blotting能检测到HRP的表达;加药及放射组比单纯放射组细胞存活分数降低,SERSF2为1.302;与A组相比,B、C、D组G2/M期细胞比例升高,S期细胞比例降低,细胞凋亡率均显著增高(P<0.01);D组与B组相比,G2/M期细胞比例升高,S期细胞比例百分比降低,凋亡率增高(P<0.01);D组与C组比较,G0/G1期、G2/M期细胞比例升高(P<0.05),S期细胞比例百分比降低,凋亡率增高(P<0.01)。结论:HRP/IAA系统与放射联合应用能更有效地抑制Hep-2细胞生长、诱导其凋亡,HRP/IAA系统具有放射增敏性。

Objective： To study the effect of Horseradish Peroxidase（HRP）/Indole-3-acetic Acid（IAA） system combined with radiation on human laryngeal squamous carcinoma cell line Hep-2. Methods： Hep-2 cells were transiently transfected with the plasmid pcDNA3.1-HRP, and the expression was detected by RT-PCR and Western blotting. The HRP/IAA system and radiation were used separately or in combination for Hep-2 cells to compare their effects. Transfected Hep- 2 cells were divided into four groups： A： control, B： 0. 5 mmol/L IAA, C： 2 Gy γ-ray, D： 0.5 mmol/L IAA combined with 2 Gy ）＇-ray. The radiosensitivity was determined by clonogenic assay, the change in radiosensitivity was quantifited by calculating the SER（sensitization enhance rate） through survival curves. Each group＇s cell-cycle change was assayed by FCM, and Annex-inV-FITC agent was used to detect cell apoptosis rate of each group. Results： The HRP expression was confirmed by RT-PCR and Western blotting. The cell survival rate of combination group was significantly lower than that of radiation group, SERSF2 for HRP/IAA was 1. 302. Compared with those of group A, cell numbers of group B, C and D in G2/M stages increased while in S stage got down （P〈0.01）. Compared with those of group B, cell numbers of group D increased in G2/M stages while decreased in S stage（P〈0.01）. Compared with group C, cell numbers of group D in G0/G1 ,G2/M stages increased while in S stage reduced （P〈0.05）. Apoptosis rate of group B,C and D increased compared with that of group A（P〈0.01）, and apoptosis rate of group D was higher than that of group B or C（both P〈0.01）. Conclusion： The results showed that the combination of HRP/IAA system with radiation may be more effective in inhibiting the growth of Hep-2 cells and inducing apoptosis. HRP/IAA system can significantly enhance the ra- diosensitivity of Hep-2 cells.