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

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。

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

Objective: To study the effect of Horseradish Peroxidase(HRP)/Indole3-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 γ-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 AnnexinV-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 radiosensitivity of Hep-2 cells.