RNA干扰环氧化酶-2抑制人表皮样喉癌细胞系增殖和侵袭的研究  

Lentivirus-mediated siRNA Targeting Cyclooxygenase-2 Gene Inhibits Human Laryngocarcinoma Cells Proliferation and Invasion

作　　者：王晓侠[1,2] 姚小宝[2] 张少强[1] 

机构地区：[1]西安交通大学医学院第一附属医院耳鼻喉科,西安710061 [2]解放军451医院耳鼻喉科

出　　处：《华西医学》2013年第2期195-199,共5页West China Medical Journal

基　　金：陕西省卫生厅科研基金(2010D40)~~

摘　　要：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。Objective To explore the effect of suppressing Cyclooxygenase-2（COX-2） overexpression in Hep-2 cells line on the proliferative rate of Hep-2 cells,in order to study a mechanism of action to find a new therapeutic method for laryngocarcinoma.Methods RT-PCR was used to detect the expression of COX-2 gene in Hep-2 cells.Lentiviral RNAi expression vector bought from Genken Company on COX-2 was constructed.After transfection into Hep-2 cells,real-time quantitation PCR was used to detect the expression alteration of COX-2,and growth curve was utilized to observe the alteration of cell growth rate.Flow cytometer was used to detect cell growth cycle.And Boyden invasion assay was used to detect the external invasion.Results COX-2 lentiviral RNAi expression vector was successfully constructed and Hep-2 cells of interfering COX-2 were built.Overexpression of COX-2 gene was greatly suppressed by detection of real-time quantitation PCR,and proliferative rate of Hep-2 cells was markedly decreased using growth curve detection after transfection of COX-2 Lentiviral RNAi expression vector into Hep-2 cells.The results of flow cytometer showed apoptosis of induced Hep-2 cells in group KD;Cell count during stage G0-G1 raised significantly,with reduced in stage S,indicating that cell proliferation was baffled.External invasion assay showed that Hep-2-AS invasion cell number（31.0 ± 1.8） was significantly lower than Hep-2 cells（104.0 ± 2.6） and Hep-2-P cells（99.0 ± 2.7）（P0.05）.Conclusion Overexpression of COX-2 gene suppressed by RNA interference reduces the proliferative rate of Hep-2 cell line,and at the same time validates that the COX-2 gene RNA interference in antitumor therapy has potential application prospects.

关 键 词：喉癌 环氧化酶-2基因 慢病毒 基因沉默 

分 类 号：R739.65[医药卫生—肿瘤]