腺病毒介导的cox-2反义RNA对食管癌细胞株DNA和蛋白质合成的影响  

Effects of adenovirus-mediated human cox-2 antisense RNA on synthesis of DNA and proteins in esophgeal carcinoma cell line

作　　者：李胜保[1] 吴清明[1] 王强[1] 王小虎[1] 谢国建[1] 

机构地区：[1]郧阳医学院附属太和医院消化内科,湖北省十堰市442000

出　　处：《世界华人消化杂志》2003年第5期517-521,共5页World Chinese Journal of Digestology

摘　　要：目的:构建表达人cox-2反义RNA的腺病毒载体,研究其对人食管癌细胞株DNA和蛋白质合成的影响.方法:把人cox-2的cDNA片段反向克隆于穿梭质粒pHCMVSP1A的CMV启动子之下,获得pAd-AShcox-2,通过脂质体与pJM17共转染293细胞,经同源重组产生编码cox-2反义RNA的重组腺病毒--Ad-AShcox-2.经PCR的方法鉴定为阳性克隆并大量扩增、纯化,转染食管癌细胞EC9706,采用生长细胞计数,免疫细胞化学、3H-TdR、3H-Leucine掺入法,研究对食管癌细胞生长、DNA及蛋白质合成的影响.结果:成功构建并扩增、纯化得到编码cox-2反义RNA的重组腺病毒Ad-AShcox-2,滴度达0.86×1012PFU/ml;Ad-AShcox-2感染肿瘤细胞后,cox-2表达水平明显降低,3H-TdR、3H-Leucine掺入量明显减少,与对照组在48h、72h、96h比较有显著性差异(q48h=16.36及16.36,q72h=39.07及19.90,q96h=54.80及30.33;P<0.001);同时发现食管癌细胞的生长明显受抑制.结论:表达cox-2反义RNA重组腺病毒感染人食管癌细胞后可降低cox-2表达水平,使DNA合成降低、蛋白质合成减少,且抑制食管癌细胞生长、增生,提示抑制cox-2的表达可能是治疗食管癌的一种新途经.AIM:To construct the recombinant adenovirus encoding human cox-2 antisense RNA, and to investigate its effect on synthesis of DNA and proteins in esophgeal carcinoma cell line EC9706. METHODS:The shuttle plasmid encoding antisense cox-2 was constructed by cloning cox-2 cDNA fragment in the reverse direction into the pHCMVSP1A. Then the plasmid pJM17 and the shuttle plasmid were cotransferred into 293 cells with lipofectamine for homologouse recombinantion to acquire recombinant adenovirus confirmed by PCR. The expressions of cox-2 in esophgeal carcinoma cell line EC9706 cells were evaluated, and its effects on cell proliferation were determined by cell growth rate, 3H-TdR and 3H-Leucine incorporation. RESULTS:The recombinant adenovirus encoding antisense cox-2 fragment ad-AShcox-2 was obtained with the titer of 0.86±1012 PFU/ml. Ad-AShcox-2 can reduce the expression of cox-2, and inhibit cell growth rate and cause cellular death. Meanwhile, The efficiencyof 3H-TdR and 3H-Leucine incor- poration was significant lower than that in the control group at 48, 72, 96 hours (q48 h = 16.36 vs 16.36 , q 72 h = 39.07 vs 19.90 , q96 h= 54.80 vs 30.33;P <0.001).CONCLUSION:Reducing the expression of cox-2 may inhibit the proliferation of esophageal cancer cells through inhibitingthe synthesis of DNA and protein.

关 键 词：腺病毒 COX-2 反义RNA 食管癌 DNA合成 蛋白质合成 

分 类 号：R735.1[医药卫生—肿瘤]