短发夹RNA对头颈肿瘤细胞端粒酶活性和增殖的影响  

Effect of Short Hairpin RNA on Telomarase Activity and Cell Viability of Head and Neck Neoplasms

作　　者：段洪刚[1] 陶泽璋[1] 陈始明[1] 肖伯奎[1] 

机构地区：[1]武汉大学人民医院耳鼻咽喉-头颈外科,武汉430060

出　　处：《武汉大学学报（医学版）》2006年第3期283-286,i0004,共5页Medical Journal of Wuhan University

基　　金：国家自然科学基金资助项目(编号:30471873)

摘　　要：目的:观察靶向人端粒酶逆转录酶(hTERT)mRNA的短发夹RNA(shRNA)对喉癌Hep-2细胞株和鼻咽癌NEC细胞株端粒酶活性和生长增殖影响,探讨抑制端粒酶活性途径治疗头颈肿瘤的可行性。方法:根据hTERT cDNA序列构建表达shRNA、含荧光素基因、靶向hTERT mRNA的真核表达质粒shRNA1和表达shR-NA的、含荧光素基因的、靶向非人类基因的真核表达质粒shRNA2。分别以质粒shRNA1、shRNA2、转染试剂及空白培养液处理体外培养的Hep-2细胞和NEC细胞。转染48 h后以TRAP PCR-ELISA法检测细胞端粒酶活性并行HE染色;倒置显微镜下每天观察细胞形态;MTT法检测细胞增殖活性。结果:①Hep-2细胞和NEC细胞shRNA1处理48 h后与空白对照组比较端粒酶活性下降,P<0.05。②HE染色发现shRNA1处理后Hep-2和NEC细胞胞体缩小、核固缩,同等培养条件下,细胞生长密度变稀,见许多圆形死亡细胞。③与相应时间点空白对照组细胞平均吸光度(A)值比较,shRNA1处理组Hep-2细胞和NEC细胞24,48,72 h A值均减小,P<0.05。结论:靶向hTERT mRNA的小干扰RNA能够显著降低Hep-2细胞和NEC细胞的端粒酶活性,抑制癌细胞生长增殖,并诱导细胞凋亡。Objective： To investigate the effect of RNA interference by targeting human telomerase reverse transcriptase （hTERT） mRNA in the larynx cancer Hep-2 cell line and nasopharyngeal carcinoma NEC cell line. Methods： The primary structures of hTERT cDNA were found in Gen- Bank. Then the structure analysis was done according to the strategy of RNA interference, by which the specific base sequences were determined to design shRNA（short hairpin RNA） plasmid. Plasmid shRNA1 involved in fluorescein gene were synthesized based on the specific base sequences. Control plasmid and shRNA2,a random sequence, were also constructed. Cells were treated daily with shRNA1, shRNA2 or only Lipofectamine. At 48 hours, the cell＇s telomerase activity was determined by the telomeric repeat amplification protocol （TRAP）-enzyme-linked immunosorbent assay （PCR-ELISA）, and the morphology alteration of the cells was observed by HE stain. At 24, 48 and 72 hours, cell viability after administration was determined respectively using the MTT assay. Results： It was observed that both Hep-2 cells and NEC cells treated with shRNA1 showed a decrease in telomerase activity compared to the other groups（all P〈0.05）. The treatment with shRNA1 in the presence of a cationic liposome decreased cell viability of Hep- 2 and NEC cells within three days （P〈0.05）. Sparse cells in culture were also observed. While none of such effects were found in any other group. Conclusion： Short hairpin RNA by targerting human telomerase reverse transcriptase （hTERT） mRNA can inhibit laryngeal carcinoma and nasopharyngeal carcinoma cells＇telomerase activity and induce cells to apoptosis.

关 键 词：端粒酶逆转录酶 短发夹RNA 头颈肿瘤 基因治疗 

分 类 号：R739.6[医药卫生—肿瘤]