RNA干扰抑制端粒酶反转录酶联合γ射线对人喉癌细胞的作用

Effect of shRNA inhibiting hTERT gene expression combined with γ-irradiation on human laryngeal cancer cells

目的观察人端粒酶反转录酶特异性干扰载体（pshRNA-hTERT）联合放射线在细胞和动物模型水平上对人喉鳞癌Hep-2细胞株的生长抑制作用，研究抑制hTERT在放射增敏中的作用。方法细胞水平：联合pshRNA-hTERT和γ射线作用于人喉癌细胞Hep-2，用TRAP-PCR，ELISA法检测端粒酶活性，彗星电泳检测DNA损伤；动物水平：建立Hep-2和Hep-2R移植瘤模型，瘤内注射pshRNA-hTERT并联合放射线观察对移植瘤的抑制作用，TUNEL法检测肿瘤细胞的凋亡，免疫组织化学法检测肿瘤内hTERT蛋白的表达。结果转染pshRNA-hTERT后Hep-2细胞hTERT的mRNA表达抑制率为60.78％；pshRNA-hTERT不仅能抑制Hep-2细胞的端粒酶活性，而且抑制照射后DNA损伤的修复；在移植瘤模型中，pshRNA-hTERT与放射线有协同抑制移植瘤生长的作用（Hep-2：EPO=1.79；Hep-2R：EPO=2.01）。结论pshRNA-hTERT在细胞和动物实验水平均具有放射增敏作用，表明抑制端粒酶及其亚单位可以增加在体肿瘤的放射敏感性，这为喉癌的基因放疗研究提供了依据。

Objective To construct an eukaryotic expression vector of human telomerase reverse transcriptase （hTERT） gene specific shRNA, and investigate the effect of pshRNA-hTERT combined with γ-irradiation on telomerase activity and DNA damage. Methods The recombinant expression plasmid pshRNA- hTERT was constructed and transfected into Hep-2 cells. The telomerase activity was examined by the PCR-based telomerie repeat amplification protocol （TRAP）. DNA single-stranded break （SSB） and the DNA double-stranded break （DSB） were detected by Comet assay. The xenograft model of human laryngeal carcinoma with the same genetic background and different radiosensitivity （Hep-2 and Hep-2R） was established in nude mice. The mixture of pshRNA-hTERT and liposome was injected to the transplanted tumor to observe the inhibition of the tumor growth. The cell apoptosis was detected by TUNEL. The hTERT protein expression was determined by streptavidin-peroxidase conjugated method （AP）. Results Recombinant expression plasmid pshRNA-hTERT was successfully constructed and transfected into Hep-2 cells. The hTERT expression inhibition rate reached 60.78%. pshRNA-hTERT not only inhibited telomerase activity of Hep-2 including the increase of telomerase activity induced by y-irradiation, but also inhibited the repair of the SSB and DSB induced by irradiation in the human laryngeal carcinoma xenograft in nude mice with the same genetic background and different radiosensitivity. The pshRNA-hTERT combined with γ-irradiation could inhibit the growth of transplanted tumor （Hep-2： EPO = 1.79; Hep-2R： EPO = 2.01） with reduced telomerase activity and hTERT protein expression. Conclusions The eukaryotic expression vector pshRNA-hTERT could enhance the radiosensitivity of Hep-2 cells in vitro and the human laryngeal carcinoma xenograft in nude mice which had the same genetic background with different radiosensitivity.