CAJ | 학술논문

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He-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.
목적 관찰인단립매반전록매특이성간우재체(pshRNA-hTERT)연합방사선재세포화동물모형수평상대인후린암Hep-2세포주적생장억제작용,연구억제hTERT재방사증민중적작용.방법 세포수평:연합pshRNA-hTERT화γ사선작용우인후암세포Hep-2,용TRAP-PCR-ELISA법검측단립매활성,혜성전영검측DNA손상;동물수평:건립Hep-2화He-2R이식류모형,류내주사pshRNA-hTERT병연합방사선관찰대이식류적억제작용,TUNEL법검측종류세포적조망,면역조직화학법검측종류내hTERT단백적표체.결과 전염pshRNA-hTERT후Hep-2세포hTERT적mRNA표체억제솔위60.78%;pshRNA-hTERT불부능억제Hep-2세포적단립매활성,이차억제조사후DNA손상적수복;재이식류모형중,pshRNA-hTERT여방사선유협동억제이식류생장적작용(Hep-2:EPO=1.79;Hep-2R:EPO=2.01).결론 pshRNA-hTERT재세포화동물실험수평균구유방사증민작용,표명억제단립매급기아단위가이증가재체종류적방사민감성,저위후암적기인방료연구제공료의거.
Objective To construct an eukaryotic expression vector of human telomerase reverso 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-hased telomeric 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 inehiding the increase of telomerase activity induced by γ-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.