γ射线增强喉癌细胞hTERTp启动子活性研究

目的 探讨γ射线对喉癌细胞中端粒酶逆转录酶基因启动子(hTERTp)活性的影响,以及通过射线增强hTERTp下游基因表达的可行性。方法 将含hTERTp的质粒转染细胞,采用报告基因评价启动子活性,通过RT-PCR和酶活性检测观察射线作用下hTERTp调控辣根过氧化物酶(HRP)的表达,克隆形成实验评价射线对phTERTp-HRP/IAA杀伤和放射增敏作用的影响。结果 在Hep-2细胞中,6 Gyγ射线照射后hTERTp活性是0 Gy照射后的2.96倍,在Hep-2R细胞中是1.60倍。质粒phTERTp-HRP转染Hep-2和Hep-2R细胞后,6 Gy照射组的HRP mRNA分别增高2.1和1.1倍,酶活性分别增高2.54和1.23倍。6 Gy联合吲哚乙酸(IAA)组Hep-2细胞的存活分数为1.3%,Hep-2R细胞的存活分数为3.5%,比对照组明显降低(F=234.280和F=357.148,P值均<0.01)。6Gy联合IAA组与IAA组相比,放射增敏比SER_<sub>SF2分别为1.52(Hep-2)和1.68(Hep-2R),存活曲线的参数α分别为0.416、0.099(Hep-2)和0.356、0.090(Hep-2R)。结论 γ射线可以增强不同放射敏感性喉癌细胞hTERTp活性,增强下游基因表达。射线联合phTERTp-HRP/IAA对喉癌细胞具有更加明显的杀伤和放射增敏作用。

Enhancement of hTERT promoter activity by γ-rays in laryngeal squamous carcinomas cells in vitro

Objective To investigate the effect of γ-rays on hTERT promoter activity in laryngeal squamous carcinomas cell lines.and to evaluate the efficiency of hTERTp mediated gene therapy combined with γ-rays.Metllods hTERTp activity was determined by luciferase assay.Plasmids phTERTp-HRP were transfected into cells.HRP expression levels were determined by RT-PCR and enzyme activity assay.The cytotoxicity and radiosensitivity of phTERTp-HRP/IAA were determined by clonogenic assay.Results After 6 Gy irradiation.a 2.96-fold increase of hTERTp activity in Hep-2 cells and a 1.60-fold increase in Hep-2Rcells were found.Hep-2 and Hep-2R cells transfected with phTERTp-HRP,HRP mRNA expression levels were 2.1-fold and 1.1-fold increased,while enzyme activity of HRP were increased by 2.54.fold and 1.23.fold.respectively after 6 Gy irradiation.Combination of 6 Gy induction and IAA incubation obviously decrease the surviving fraction of Hep-2 and Hep-2R cells(P＜0.01),and clearly radiosensitize the cells without induction(SERSF2=1.52 in Hep-2;SERSF2=1.68 in Hep-2R),the parameter α with or without 6 Gy induction before IAA incubation were 0.416,0.099(Hep-2)and 0.356,0.090(Hep-2R),respectively.Conclusions γ-rays can enhance hTERT promoter activity and impmve the expression of downstream gene in difierent radiosensitivelaryngeal squamous carcinomas cells.Combination of radiation and hTERTp-HRP/IAA construct results in significant cytotoxicity and radiosensitization.