目的 构建沉默环氧化酶-2（COX-2）基因重组慢病毒，观察其体外侵袭的抑制作用，从而探讨干扰COX-2抑制喉癌细胞增殖的作用机理，为喉癌的治疗提供新的思路。 方法 逆转录聚合酶链反应（RT-PCR）检测COX-2基因在人表皮样喉癌细胞（Hep-2）中的表达情况。利用上海吉凯公司RNA干扰（RNAi）慢病毒表达载体系统，构建针对COX-2基因慢病毒RNAi表达载体。转染Hep-2细胞，干扰COX-2基因的表达，实时定量PCR检测干扰前后基因表达变化。利用生长曲线测定干扰载体转染前后细胞生长速度变化。流式细胞仪检测细胞的生长周期。Boyden侵袭小室法测定体外侵袭力。 结果 成功构建了COX-2慢病毒RNAi表达载体，并建立了干扰COX-2基因的Hep-2细胞系。实时定量PCR检测COX-2基因在Hep-2细胞系中过表达被显著抑制。生长曲线测定，COX-2基因干扰后细胞增殖明显变慢。流式细胞仪检测细胞的生长周期可见干扰组诱导Hep-2细胞凋亡，转染G0～G1期细胞数量明显上升，S期细胞减少，表明siRNA干扰Hep-2细胞后，细胞由G0～G1期进入到S期受到阻滞，细胞增殖速度下降。体外侵袭实验中，Hep-2-AS侵袭细胞数（31.0 ± 1.8）显著低于Hep-2细胞（104.0 ± 2.6）及Hep-2-P细胞（99.0 ± 2.7），差异有统计学意义（P＜0.05）。 结论 喉癌中过表达的COX-2基因被干扰后表达明显降低并显著抑制细胞的生长速度和侵袭能力。同时验证了COX-2基因RNA干扰在进行抗肿瘤的治疗中潜在的应用前景。
【摘要】 目的 探讨喉癌组织中 EGFL7基因 mRNA表达与喉癌分化、转移及临床预后的关系。 方法 收集2008年5—11月共42例行喉癌手术患者的切除标本。用免疫组织化学法检测EGFL7蛋白在42例喉癌组织和配对癌旁组织中的表达情况，提取肿瘤及癌旁组织配对标本的总 RNA，用逆转录（RT）-PCR方法测定EGFL7基因表达，蛋白质印迹法测定EGFL7蛋白的表达，并结合临床资料，对 EGFL7基因差异表达与喉癌患者临床表现的相关性进行分析研究。 结果 对42例配对标本分别进行 EGFL7 mRNA荧光检测比较，30例标本的肿瘤组织中EGFL7基因mRNA表达明显高于癌旁正常喉组织。22例标本的肿瘤组织中EGFL7蛋白表达明显高于癌旁正常喉组织。EGFL7 mRNA的表达与喉癌淋巴结转移和浸润深度密切相关（Plt;0.05），而与患者的性别、年龄、吸烟、肿瘤分化程度等无关（Pgt;0.05）。 结论 EGFL7基因在喉癌组织中表达状态与喉癌的生长和浸润转移关系密切，EGFL7基因可望作为喉癌病情发展及指导临床治疗的标记物之一。【Abstract】 Objective To investigate the association of EGF-like-domain, multiple 7 (EGFL7) mRNA expression level with the differentiation, metastasis and prognosis of laryngocarcinoma. Methods Tissue specimens were obtained from 42 patients undergoing surgery for laryngocarcinoma between May and November, 2008. Immunohistochemistry was used to detect the level of EGFL7 in the 42 tumor tissues and matched adjacent normal tissues. Reverse transcriptional PCR (RT-PCR) was performed for amplification of EGFL7 mRNA from the 42 tumor tissues and matched adjacent normal tissues, and westerblot was adopted to determine EGFL7 protein expression. The differential EGFL7 mRNA expression was analyzed for its association with the clinical manifestations of the patients. Results EGFL7 mRNA expression was detected in all the laryngocarcinoma tissues and normal tissues adjacent to the carcinoma using fluorescence method. EGFL7 mRNA expression was significantly higher in the tumor tissues than in the adjacent tissues in 30 cases, and EGFL7 protein expression was also significantly higher in the tumor tissues than in the adjacent normal laryngeal tissues in 22 cases. Expression of EGFL7 mRNA was highly correlated with lymph node metastasis and T classification (Plt;0.05), but was not correlated with the patients’ gender, age, or tumor differentiation (Pgt;0.05). Conclusions EGFL7 mRNA expression is correlated closely with the differentiation and metastasis of laryngocarcinoma. EGFL7 may serve as a marker for assessing the progression of laryngocarcinoma and provide assistance for clinical therapeutic decisions.