喉癌细胞hTERT启动子介导基因治疗研究

hTERT promoter mediating gene therapy in laryngeal squamous carcinomas cells in vitro

目的研究喉癌细胞中端粒酶活性、端粒酶逆转录酶（hTERT）表达和hTERT启动子活性的关系，以及hTERT启动子介导喉癌基因治疗的可行性。方法将含hTERT启动子的质粒转染不同放射敏感性喉癌细胞（Hep2和Hep2R），通过报告基因评价启动子活性，RT-PCR检测hTERT mRNA表达，PCR—ELISA法检测端粒酶活性。构建质粒phTERTp-HRP，用RT-PCR和酶活性分析评价辣根过氧化物酶（HRP）表达，以克隆形成实验评价phTERTp-HRP／吲哚乙酸（IAA）对克隆形成率和放射敏感性的影响。结果Hep2R的端粒酶活性、hTERT mRNA表达水平和hTERT启动子活性分别是Hep2的1．37、1．43和1．81倍。hTERT启动子活性与hTERT mRNA表达水平和端粒酶活性之间显著正相关（P〈0．01）。转染phTERTp-HRP后，Hep2R细胞中HRP mRNA表达和HRP活性水平分别是Hep2细胞的2．1倍和1．8倍。0．5mmol／L IAA处理后，SERSF2分别为1．24（Hep2R）和1．20（Hep2），无IAA和有IAA组存活曲线参数α分别为0．020、0．090（Hep2R）和0．042、0．099（Hep2）。结论hTERT启动子可用于不同放射敏感性喉癌细胞的基因治疗。hTERTp—HRP／IAA基因治疗有望用于喉癌细胞的靶向杀伤和放射增敏。

Objective To investigate the relationship among hTERT promoter activity, hTERT mRNA expression, and telomerase activity （TA） in laryngeal squamous carcinomas cell lines, and to evaluate the usefulness of hTERT promoter mediated gene therapy. Methods After plasmids pGL3-hTERTp were transfected, hTERT promoter activity, hTERT mRNA expression and TA were determined by luciferase assay, RT-PCR and TRAP-PCR-ELISA, respectively. Plasmid phTERTp-HRP was constructed and transfected, HRP expression was determined by RT-PCR and competent peroxidase activity was confirmed by enzyme activity assay. The cytotoxicity and radiosensitivity of phTERTp-HRP/IAA were determined by clonogenic assay. Results The relative levels of hTERT promoter activity, hTERT mRNA expression and TA in Hep2R cells were 1.37-fold, 1.43-fold and 1.81-fold compared with Hep2R cells, hTERT promoter activity was closely associated with hTERT mRNA expression and TA levels（ P 〈 0.01 ）. In phTERTp-HRP transfected cells, the HRP mRNA expression level and HRP activity in Hep2R cells were 2.1-fold and 1.8-fold compared with Hep2R cells. After IAA incubation, the sensitizer enhancement ratio （ SERsF2 ） was 1.24 （ Hep2R cells） and 1.20 （ Hep 2cells） , the parameter a of with or without IAA incubation were 0.090, 0.020 （Hep2R） and 0.099, 0.042 （Hep2）. Conclusions hTERT promoter is applicable in mediating gene therapy in different radiosensitive laryngeal squamous carcinomas cells, hTERTp-HRP/IAA gene therapy may be a promising supplementary method for radiotherapy of laryngeal squamous-cell carcinomas.