摘要
目的:探讨DNA载体途径的RNA干扰技术抑制人端粒酶逆转录酶(hTERT)基因表达及诱导喉癌细胞Hep-2凋亡的作用。方法:构建靶向hTERTmRNA的质粒pshRNA1、pshRNA2及对照质粒pshRNA3、pEGFP,并将其分别转染喉鳞癌Hep-2细胞。共聚焦荧光显微镜观察质粒转染及表达情况;RT-PCR测定hTERTmRNA表达;Western印迹测定hTERT蛋白表达;末端重复片断扩增-酶联免疫吸附(TRAP-ELISA)方法测定细胞端粒酶活性;MTT法研究细胞增殖活性变化;原位细胞凋亡(TUNEL)及透射电子显微镜研究细胞凋亡。结果:①共聚焦荧光显微镜下见大量的细胞呈现绿色荧光,与其他组比较,pshRNA1、2组呈现荧光的死亡细胞显著增加。②pshRNA1、2转染细胞后hTERTmRNA及hTERT蛋白表达显著降低;其端粒酶活性明显受到抑制:转染后2 d pshRNA1组活性为:0.159±0.039、pshRNA2组活性为0.163±0.028,与空白对照组1.523±0.076比较,差异有非常显著性(P<0.005)。③pshRNA1、2转染细胞后可显著抑制细胞增殖、诱导细胞凋亡。这两组细胞透射电镜可见典型细胞凋亡特征。结论:DNA载体途径的RNA干扰技术能有效抑制hTERT基因的表达及端粒酶活性并诱导癌细胞凋亡,此法可能成为抑制肿瘤细胞的新途径。
Objective：To suppress human telomerase reverse transcriptase （hTERT） expression an vector-based RNA interference d induce apoptosis of laryngeal carcinoma Hep-2 cells by using DNA（RNAi） technology. Methods： Short hairpin RNA expression vectors targeting the mRNA of hTERT （pshRNA1 and pshRNA2） and the control vector （pshRNA3 and pEGFP） were constructed and then transfected into laryngeal carcinoma Hep-2 cells. Plasmid transfection and gene expression were observed by confocal laser microscopy. The mRNA expression and protein expression of hTERT were determined by reverse-trans criptase polymerase chain reaction and western blotting, respectively. The activity of telomerase was measured by telomeric repeated amplification enzyme-linked immunosorbent assay （TRAP-ELISA）. Cell viability was evaluated by 3-（4,5-dimethyl thiazol-2-yl） 2,5-diphenyl tetrazolium bromide （MTT） assay. Cell apoptosis was detected by TUNEL staining and transmission electron microscope. Results.. ① The strong intensity of green fluorescence was observed indicating there were many dead cells in pshRNA1 and pshRNA2 groups than that in control group. ②hTERT mRNA and protein expressions in pshRNA1 and pshRNA2 groups significantly decreased. The activity of telomerase in these two groups were markedly inhibited （pshRNA1 ： 0.159 ± 0.039; pshRNA2： 0.163±0. 028, control： 1. 523±0.076. P〈0.005）. ③Transfections of Hep-2 cells with pshRNA1 and pshRNA2 significantly inhibited proliferation and induced apoptosis of Hep-2 cells. The cells in two groups above showed typical apoptotic characteristics. Conclusion.. The expression of hTERT and the activity of telomerase were efficiently suppressed by using DNA vector-based RNA interfering technology. It can be a new strategy for the treatment of laryngeal carcinoma.
出处
《肿瘤》
CAS
CSCD
北大核心
2006年第9期798-804,共7页
Tumor
基金
国家自然科学基金资助项目(编号:30471873)
