目的 構建沉默環氧化酶-2（COX-2）基因重組慢病毒，觀察其體外侵襲的抑制作用，從而探討干擾COX-2抑制喉癌細胞增殖的作用機理，為喉癌的治療提供新的思路。 方法 逆轉錄聚合酶鏈反應（RT-PCR）檢測COX-2基因在人表皮樣喉癌細胞（Hep-2）中的表達情況。利用上海吉凱公司RNA干擾（RNAi）慢病毒表達載體系統，構建針對COX-2基因慢病毒RNAi表達載體。轉染Hep-2細胞，干擾COX-2基因的表達，實時定量PCR檢測干擾前后基因表達變化。利用生長曲線測定干擾載體轉染前后細胞生長速度變化。流式細胞儀檢測細胞的生長周期。Boyden侵襲小室法測定體外侵襲力。 結果 成功構建了COX-2慢病毒RNAi表達載體，并建立了干擾COX-2基因的Hep-2細胞系。實時定量PCR檢測COX-2基因在Hep-2細胞系中過表達被顯著抑制。生長曲線測定，COX-2基因干擾后細胞增殖明顯變慢。流式細胞儀檢測細胞的生長周期可見干擾組誘導Hep-2細胞凋亡，轉染G0～G1期細胞數量明顯上升，S期細胞減少，表明siRNA干擾Hep-2細胞后，細胞由G0～G1期進入到S期受到阻滯，細胞增殖速度下降。體外侵襲實驗中，Hep-2-AS侵襲細胞數（31.0 ± 1.8）顯著低于Hep-2細胞（104.0 ± 2.6）及Hep-2-P細胞（99.0 ± 2.7），差異有統計學意義（P＜0.05）。 結論 喉癌中過表達的COX-2基因被干擾后表達明顯降低并顯著抑制細胞的生長速度和侵襲能力。同時驗證了COX-2基因RNA干擾在進行抗腫瘤的治療中潛在的應用前景。
【摘要】 目的 探討喉癌組織中 EGFL7基因 mRNA表達與喉癌分化、轉移及臨床預后的關系。 方法 收集2008年5—11月共42例行喉癌手術患者的切除標本。用免疫組織化學法檢測EGFL7蛋白在42例喉癌組織和配對癌旁組織中的表達情況，提取腫瘤及癌旁組織配對標本的總 RNA，用逆轉錄（RT）-PCR方法測定EGFL7基因表達，蛋白質印跡法測定EGFL7蛋白的表達，并結合臨床資料，對 EGFL7基因差異表達與喉癌患者臨床表現的相關性進行分析研究。 結果 對42例配對標本分別進行 EGFL7 mRNA熒光檢測比較，30例標本的腫瘤組織中EGFL7基因mRNA表達明顯高于癌旁正常喉組織。22例標本的腫瘤組織中EGFL7蛋白表達明顯高于癌旁正常喉組織。EGFL7 mRNA的表達與喉癌淋巴結轉移和浸潤深度密切相關（Plt;0.05），而與患者的性別、年齡、吸煙、腫瘤分化程度等無關（Pgt;0.05）。 結論 EGFL7基因在喉癌組織中表達狀態與喉癌的生長和浸潤轉移關系密切，EGFL7基因可望作為喉癌病情發展及指導臨床治療的標記物之一。【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.