中文摘要：

目的 探讨RNA干扰抑制端粒酶逆转录酶（human telomerase revese transcriptase，hTERT）对喉癌Hep-2细胞凋亡、P53及Caspase-3蛋白表达的影响。方法 根据hTERT cDNA序列构建表达hTERT mRNA特异的、含荧光素基因的shRNA真核表达质粒pshRNA1，随机选取一段与人类基因无同源性的碱基序列构建表达shRNA的含荧光素基因的对照质粒pshRNA2，采用METAFECTENE作为转染试剂。分别以质粒pshRNA1、pshRNA2及空白培养液转染喉癌Hep-2细胞。电镜下观测细胞凋亡小体的形成，TUNEL法检测细胞的原位凋亡，Western Blot法检测hTERT、P53、Caspase-3蛋白表达水平。结果 pshRNA1转染组hTERT蛋白表达显著下调，大量Hep-2细胞凋亡，电镜显示凋亡小体形成。pshRNA1转染组P53和Caspase-3蛋白表达水平显著高于其他处理组及空白对照组（P〈0．001），二者升高趋势具有相关性（相关系数r=0．088，P〈0．05）。结论 RNAi抑制hTERT基因表达能有效诱导喉癌细胞凋亡，其可能的分子机制是诱导P53及Caspase-3蛋白表达上调。

英文摘要：

Objective To investigate the effect of inhibiting human telomerase reverse transcriptase （hTERT） on apoptosis and expression of P53, Caspase-3 proteins by RNA interference （RNAi） in the laryngeal cancer cell line, Hep- 2 Methods The primary structures of hTERT cDNA were found in GeneBank. Then the structure analyses were done according to the strategy of RNAi, which determined the specific base sequences to design shRNA plasmid. One type of plasmid, including fluorescein gene, pshRNA1 was synthesized based on the specific base sequence. A random sequence, pshRNA2 was also constructed as control. METAFECTENE was used as the transfection reagent. Cells were treated daily with pshRNA1-2 or normal culture medium respectively. After administration of pshRNA1-2, apoptotic Hep-2 cells were detected by TUNEL method and transmission electron microscope respectively, hTERT, P53 and Caspase-3 proteins were examined by Western Blot. Results After administration of pshRNA1 for up to 24 hours or 48 hours, the mean apoptotic cell numbers were 32.30 %,6 and 34.52 % （ P 〈 0.01）, and the apoptotic characteristic was abserved with electron microscope. The expression of hTERT protein, significantly decreased after treated by pshRNA1 （ P 〈 0.05）. The expression of P53 and Caspase-3 proteins both significantly increased after treated by pshRNA1 （ P 〈0.001）, and there was a eorrelationship between these proteins （ r = 0. 088, P 〈 0.05）. Conduslon The RNAi targeting against hTERT induces apoptosis of Hep-2 cells by increasing the expression of P53 and Caspase-3 proteins.