摘要
目的 观察鼻咽癌细胞中的RNA干扰效应，确定hTERT基因RNA干扰的有效作用位点，探讨RNA干扰在鼻咽癌基因治疗中的应用前景。方法 利用体外合成法合成针对hTERT基因的小干扰RNA（siRNA）序列，脂质体转染鼻咽癌CNE-2细胞，逆转录聚合酶链反应（reverse transcriptase polymemse chain reaction，RT-PCR）检测转染细胞中hTERT基因的mRNA水平，Western Blot观察siRNA干扰后hTERT蛋白表达水平，噻唑蓝法检测siRNA对细胞生长抑制作用，流式细胞学检测siRNA诱导细胞凋亡的作用。结果 在siRNA转染CNE-2细胞后，hTERT的mRNA表达水平分别下调10％～70％，蛋白表达水平不同程度降低，细胞周期在G0～G1期受阻，并不同程度诱导细胞凋亡。结论 siRNA可在鼻咽癌中有效发挥RNA干扰效应，下调hTERT基因mRNA及蛋白表达水平，降低端粒酶活性，抑制鼻咽癌细胞增殖并诱导其凋亡，为鼻咽癌基因治疗研究奠定了一定的基础。
Objective To explore the effect of RNA interference （RNAi） on nasopharyngeal carcinoma cells, the effective positions in hTERT gene for RNAi, and to discuss the future application of RNAi in the gene therapy of nasopharyngeal carcinorma. Methods Small interfering RNAs （siRNA） targeting hTERT gene was synthesized by Turboscript^TM T7 Transcription kit, and siRNA was transfected into CNE-2 cells by using GeneSilencer^TM siRNA transfection reagent. The hTERT mRNA and hTERT ptotein were detected by reverse transcriptase polymerase chain reaction （RT-PCR） and Western blotting respectively. Thiazolyl blue （MTT） assay was used to determine the inhibitory effect of siRNA on cell growth and flow cytometry was used to observe the apoptosis induced by siRNA. Results After the transfection of CNE-2 cells, the expressions of hTERT mRNA and hTERT protein were down-regulated at different degree Meanwhile, the cell cycle was suffocated at G0-G1 stage and cell apoptosis was induced. Conclusion The synthesized siRNA can effectively interfere nasopharyngeal carcinoma cells by down-regulation the expressions of the hTERT mRNA and its protein, therefore can inhibit the growth of the cells and induce their apoptosis. Future application of RNAi in the gene therapy of nasopharyngeal carcinoma might be expected.
出处
《中国耳鼻咽喉颅底外科杂志》
CAS
2006年第2期91-95,共5页
Chinese Journal of Otorhinolaryngology-skull Base Surgery
基金
国家自然科学基金资助项目(30471875)
