A number of different approaches have been developed to inhibit telomerase activity in human cancer cells. In this study, the effect of antisense oligonucleotides (ODNs) by targeting human telomerase reverse transcriptase (hTERT) mRNA in a laryngeal cancer cell line (Hep-2) was investigated.A 20mer antisense oligodeoxynucleotide targeting the most open part of hTERT mRNA (anti-hTERT) and a mismatched control sequence were synthesized. Cells were treated daily with oligonucleotides for up to 72 hours. hTERT mRNA expression was measured by the reverse transcription polymerase chain reaction (RT-PCR) assay; telomerase activity by the telomerase PCR ELISA assay kit (TRAP; Boehringer Mannheim, GmbH, Mannheim, Germany). Cell viability after administration of ODNs was determined using the MTT assay. Morphological changes were examined by haematoxylin and eosin staining.The cell cycle was analyzed using flow cytometry. It was found that antisense treatment induced a decrease in hTERT mRNA expression, telomerase activity, cell growth rate, cell viability, and an increase in apoptosis. The results suggest that inhibition of telomerase activity in Hep-2 cells by short-term antisense treatment against the mRNA of hTERT results in apoptotic cell death. The treatment with anti-hTERT may be useful as a treatment modality for laryngeal squamous carcinoma.

Objective: Telomerase activity is mainly regulated by the human telomerase reverse transcriptase (hTERT) gene. Our objective was to investigate the effect of short hairpin RNA (shRNA) on hTERT expression and telomerase activity in laryngeal cancer cells. Design: Short hairpin RNA expression vectors targeting the messenger RNA of hTERT were constructed. Cells were treated with shRNA expression vectors directed against 2 different hTERT sites, control vectors that included mismatched shRNA and those without shRNA. The expression of hTERT was determined by rsetranscriptase polymerase chain reaction and Western blotting. The activity of telomerase was measured by telomeric repeated amplification enzyme-linked immunosorbent assay. The cell viability was examined using the 3-(4,5-dimethyl thizol-2-yl) 2,5-diphenyl tetrazolium bromide assay. Results: We found that treatment of shRNA expression vectors induced a significant decrease in hTERT messenger RNA expression, the level of hTERT protein, telomerase activity, and cell viability. All of these effects were seen regardless of the target site, and the shRNA control showed none of these effects. Conclusion: Our results suggest that shRNA directed against hTERT inhibits telomerase activity through suppression of the hTERT expression in laryngeal cancer cells and that RNA interfering technology may be a promising strategy for the treatment of laryngeal cancers.

目的：探讨RNA干扰hTERT（人端粒酶逆转录酶）基因对人喉鳞状细胞癌的治疗作用。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pshRNA1、pshRNA2。将shRNA质粒分别转染人喉癌Hep-2细胞株及荷瘤裸鼠瘤体内。以激光共聚焦显微镜观察质粒在Hep-2细胞及瘤体内的表达；以MTT法观察质粒对Hep-2细胞增殖的抑制作用；以蛋白印迹法（Western blot法）检测Hep-2细胞中hTERT蛋白的表达；以免疫组化SP 法检测裸鼠瘤体内hTERT蛋白的表达。结果：pshRNA1、pshRNA2转染Hep-2细胞及pshRNA1转染瘤体后，共聚焦显微镜下见大量的癌细胞表达绿色荧光，hTERT蛋白表达明显下降。细胞受pshRNA1、pshRNA2转染后, 其生长活性受到明显抑制。体内抑瘤实验表明，与空质粒载体组（pshRNA4）和生理盐水组相比，pshRNA1 组移植瘤生长明显受到抑制。结论：表达shRNA的、hTERT基因特异的真核表达质粒能有效转染体内、外喉鳞癌细胞，并可有效抑制人喉鳞癌细胞的生长，为今后应用RNA干扰基因治疗喉癌提供重要的实验参考。

目的：探讨应用RNA干扰技术抑制Hep-2细胞人端粒酶逆转录酶（hTERT）基因对C-myc蛋白表达的影响。方法：根据hTERT eDNA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及空白培养液处理喉癌Hew2细胞。应用RT-PCR法检测hTERT基因的表达，Western Blot法检测hTERT和C-myc蛋白表达水平。结果：质粒pshRNA1转染组hTERT mRNA及蛋白表达水平显著低于其他处理组及空白对照组（P<O.05）；质粒pshRNA1转染组C-myc蛋白表达水平显著高于其他处理组及空白对照组（P<O.01）。结论：RNA 干扰抑制人端粒酶逆转录酶活性使喉癌Hep-2细胞C-myc蛋白表达升高，其确切的机制有待进一步研究。

目的：探讨核因子κB/p65（NF-κB/p65）和环氧合酶2（COX-2）在喉鳞状细胞癌（LSCC）中的表达和临床意义及两者间可能存在的相互关系。方法：采用免疫组织化学SP法检测41例LSCC和10例声带息肉组织中NF-κB/p65和COX-2蛋白的表达水平。结果：NF-κB/p65和COX-2蛋白在喉癌组与对照的声带息肉组比较有显著性差异。进一步定性分析发现COX-2与喉癌的组织病理学分化程度有关。Spearman相关分析显示NF-κB/p65与COX-2高度相关。结论：①喉癌中NF-κB/p65与COX-2的表达均升高。②NF-κB/p65可能促使COX-2的表达。③NF-κB/p65可能成为喉癌治疗的新靶点。

目的　探讨RNA干扰人端粒酶逆转录酶（hTERT）基因对Hep-2细胞生长增殖的抑制作用及诱导凋亡作用。方法　根据hTERT cDNA序列构建表达hTERTmRNA特异的、含荧光素基因的shRNA真核表达质粒p shRNA1、p shRNA2。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的含荧光素基因的质粒p shRNA3。构建不表达shRNA的含荧光素基因的对照质粒p shRNA4。实验分为5组：A（p shRNA1）、B（p shRNA2）、C（p shRNA3）、D（p shRNA4）、E （空白培养液）。酶切后电泳分析鉴定质粒p shRNA1～3。采用共聚焦显微镜检测质粒转染后细胞荧光表达情况；以蛋白印迹法研究hTERT表达变化；以TRAP2PCR EL ISA 法研究端粒酶活性变化；以四甲基偶氮唑盐（MTT）法、倒置相差显微镜及原位细胞凋亡法观察各质粒抑制细胞增殖及诱导凋亡作用。结果（1）质粒p shRNA1～3 SalⅠ酶切后电泳见400 bp小带,与预期插入的目的基因大小一致。共聚焦显微镜下见大量的细胞表达绿色荧光。（2） p shRNA1、2转染细胞后hTERT表达显著降低;细胞端粒酶活性明显受到抑制,A组细胞活性为: 01159 ±01039、B组细胞活性为01163 ±01028、E组细胞活性为11512 ±01076。A、B组与E组比较,差异均有统计学意义（P < 0101） 。（3）与E组细胞吸光度（A ）值比较,A、B组细胞各时间点均减小, P值均小于0101。同等培养条件下,A、B组脱落瓶壁的死亡细胞显著增多,凋亡率明显升高。结论　（1）靶向hTERTmRNA的shRNA真核表达质粒能有效转染Hep22细胞; （2） RNA干扰hTERT能显著地抑制Hep22细胞的生长增殖并诱导细胞凋亡。

目的　探讨鼻中隔偏曲患者两侧窦口鼻道复合体（ostiomeatal complex，OMC）的解剖变异。方法　对103例不同部位鼻中隔偏曲的患者作鼻内窥镜检查和鼻窦冠状位、轴位CT扫描，比较两侧OMC解剖变异发生率和鼻窦炎发病率的差异。结果　偏曲对侧中、下鼻甲肥大的发生率明显高于同侧（P<0.05），筛泡和鼻丘气房的宽度也明显大于同侧（配对t检验，P<0.01）；中鼻甲反常曲线偏曲同侧的发生率明显高于对侧（配对t检验，P<0.01），其它解剖变异偏曲两侧差异无显著性。偏曲两侧鼻窦炎的发病率差异无显著性（P>0.05）。结论　鼻中隔偏曲可能引起偏曲对侧中鼻甲及鼻腔外侧壁结构的代偿性改变，这种改变可能在偏曲对侧鼻窦炎的发病中起着一定的作用。

Paranasal sinuses chordoma is rare in pediatric patients.We report a case of a 15-month-old patient who presented with a nasal mass, accompanied by gradual nasal obstruction since 3 months of age. A 3.2cm

目的 探讨端粒酶逆转录酶（telomerase reverse transcriptase，TERT）和核因子KB/p65（NF-B/p65）在喉鳞状细胞癌中的表达及两者间可能存在的相互关系。方法采用免疫组化SP法检测41例喉鳞状细胞癌和1O例声带息肉组织中TERT和NF-KB/p65的表达水平。结果TERT和NF-KB/p65在喉鳞状细胞癌中的阳性表达率分别为78.O5％和68.29％，声带息肉中几乎未见TERT和NF-KB/p65的表达。定性分析发现TERT与喉鳞状细胞癌的T分级、淋巴结转移和肿瘤临床分期有关。相关分析显示NF-KB/P65与TERT呈正相关。结论①TERT和NF—KB/p65参与了喉癌的发展过程；②TERT有望作为防止喉鳞状细胞癌进一步恶化的治疗靶点；③喉鳞状细胞癌中NF-KB/p65促进了TERT的表达。

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因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 hairpinRNA，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亡，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、脾及造血系统无明显的毒副作用。