中文摘要：

背景与目的：RNA干扰（RNA interference，RNAi）是指双链RNA导入细胞后诱导靶mRNA发生特异性的降解，导致基因转录后沉默的现象。RNAi在基因功能和抗病毒基凶治疗等方面均有报道。但对喉癌等头颈恶性肿瘤中高表达的人端粒酶逆转录酶（human telomerase reverse transcriptase，hTERT）基因，目前尚未见报道。本课题利用RNAi技术，研究短发夹RNA（short hairpin RNA，shRNA）抑制hTERT基因表达对裸鼠喉鳞状细胞癌皮下移植模型的抑瘤作用。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pshRNA。建立人喉癌Hep-2细胞株裸鼠皮下接种模型。将pshRNA转染入荷瘤裸鼠瘤体内，观察肿瘤生长情况。以激光共聚焦显微镜观察质粒在瘤组织内的表达：以HE染色法观察质粒治疗后瘤组织的病理改变：以原位末端标记法（TUNEL法）检测肿瘤细胞的凋亡情况：以免疫组化SP法检测hTERT蛋白在肿瘤内的表达：光镜下观察心、肝、肾、脾结构的改变并测量血液学和血液生化指标。结果：pshRNA组（A组）、空质粒载体组（B组）与生理盐水组（C组）之间比较，A组与C组瘤体积有显著性差异（P〈0．01），B组与C组之间瘤体积无显著性差异（P〉0．05）．抑瘤率为76．50％。pshRNA及空质粒载体转染入瘤体后．共聚焦显微镜下见大量的癌细胞表达绿色荧光。病理学检查及TUNEL检测发现：A组肿瘤生长受到抑制，细胞分裂相少见．可见大量肿瘤细胞坏死及凋广，该组肿瘤细胞的凋亡指数[（26．47±4．25）％]明显高于B组[（3．40±1．41）％]和C组[（2．73±1．35）％]。给予pshRNA后瘤体内hTERT蛋白表达明显下调，而且对心、肝、肾、脾无明显损害，对造血系统无影响。结论：表达shRNA的DNA载体质粒沉默hTERT基因可显著抑制人喉癌裸鼠肿瘤的生长，而且对心、肝、肾、脾及造?

英文摘要：

BACKGROUND ＆ OBJECTIVE. RNA interference （RNAi） is triggered by the presence of double-stranded RNA （dsRNA） in the cell and results in rapid destruction and post-transcriptional gene silencing of target mRNA. The roles of RNAi in gene function and antiviral therapy have been reported, but its effect on human telomerase reverse transcriptase （hTERT） gene, which is highly expressed in laryngeal squamous cell carcinoma and other head and neck neoplasms, has seldom been reported. This study was to explore the inhibitory effect of silencing hTERT gene by short hairpin RNA （shRNA） on growth of laryngeal squamous cell carcinoma xenograft in nude mice with RNAi technique. METHODS： Plasmid pshRNA containing fluorescein gene and hTERT cDNA sequences was synthesized. Human laryngeal squamous cell carcinoma Hep-2 cells were transplanted into nude mice to establish xenograft tumors, pshRNA was transfected into the tumors. The tumor volume was observed. Fluorescence expression in the tumors was observed by laser confocal microscopy. Tumor morphology was observed with HE staining. Cell apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling （TUNEL） assay. The expression of hTERT protein in the tumors was detected by SP immunohistochemistry. The structural change of heart, liver, kidney and spleen was observed, and peripheral blood hematological and biochemical parameters were determined. RESULTS： The tumor volume was significantly smaller in pshRNA group than in normal saline （NS） group and blank plasmid group （P〈0.01）. The inhibition rate was 76.50% in pshRNA group. After transfection of pshRNA or blank plasmid, many green fluorescent cells were observed under confocal microscope. The necrosis and apoptosis of tumor cells were found under light microscope in shRNA group. The apoptotic index of tumor cells was significantly higher in pshRNA group than in NS group and blank plasmid group [（26.47±4.25）% vs. （2.73±1.35）% and （3.40±1.41）%, P?