构建人 COX-2反义核酸真核表达载体对膀胱癌 T24细胞株COX-2表达的作用

Effect of Human Antisense Nucleic Acid of Cyclooxygenase-2 （COX-2） on Expression of COX-2 in Bladder Cancer T24 Cells

【目的】构建人环氧化酶-2（COX-2）反义核酸真核表达载体，探讨其抑制膀胱癌 T24细胞COX-2表达的作用。【方法】经PCR法从含人COX-2基因的质粒中扩增其cDNA ，通过限制性内切酶克隆于 T载体EcoR Ⅰ和Xba Ⅰ位点之间，并将其反向插入pcDNA3．1中获得重组质粒。应用脂质体介导将其导入T24膀胱癌细胞中，经过G418筛后进行流式细胞术、间接免疫荧光染色和RT-PCR鉴定。【结果】核酸测序和限制性内切酶消化证实重组COX-2反义核酸真核表达质粒 pcDNA3．1/COX-2 as是正确的，经鉴定转导 pcD-NA3/COX-2 as的T24细胞中环氧化酶-2的表达明显抑制。【结论】成功构建人COX-2反义核酸真核表达载体并获得环氧化酶-2基因表达持续下调的膀胱癌细胞株T24-AS。

[Objective]To construct the antisense nucleic acid eukaryotic expression vector of human cyclooxygenase-2（COX-2） ,and to explore its role in inhibiting the expression of COX-2 in human bladder cancer T24 cells .[Methods] PCR method was used to amplify cDNA from COX-2 plasmid .The COX-2 cDNA was cloned into the sites between T vectors EcoR 1 and Xba I through restrictive endonuclease ,and then inversely inserted into pcDNA3 .1 to obtain the recombinant plasmid .The recombinant plasmid was transfected into bladder cancer T24 cells mediated by liposome .After the scanning by G418 ,flow cytometry ,indirect immuno-fluorescence staining and RT-PCR were performed .[Results] Nucleic acid and restrictive endonuclease digestion confirmed that COX-2-antisense nucleic acid eukaryotic expression plasmid pcDNA 3 .1/COX-2 was correct .Identification showed that the expression of COX-2 in T24 cells transfected with pcDNA3/COX-2 was inhibited obviously .[Conclusion] Antisense nucleic acid eukaryotic expression vector of COX-2 is constructed successfully .Bladder cancer cell line T24-AS with persistent downregulation of COX-2 expression is obtained .