摘要
目的探讨环氧合酶-2(COX-2)特异性小干扰RNA(siRNA)真核表达质粒对人结肠癌HT-29细胞COX-2表达和生长的影响。方法设计短发夹结构的COX-2 siRNA对应模版DNA序列,构建重组质粒pshCOX-2。将重组质粒转染人结肠癌HT-29细胞,采用逆转录-聚合酶链反应(RT-PCR)、Western印迹分别从mRNA和蛋白水平检测COX-2表达。用四氮唑盐(MTT)法和流式细胞仪观察COX-2表达被抑制后细胞的生长情况,放射免疫法(RIA)和ELISA法分别检测培养上清中PGE2和VEGF的含量变化。结果酶切及测序证实质粒pshCOX-2构建成功。转染重组质粒72h后可以显著抑制HT-29细胞COX-2 mRNA和蛋白表达(P<0.05),细胞生长受抑,凋亡细胞数显著增加(P<0.05),细胞PGE2和VEGF的产生受抑制(P<0.05)。结论成功构建的COX-2 siRNA真核表达质粒pshCOX-2通过抑制人结肠癌HT-29细胞COX-2基因表达,从而抑制细胞生长、PGE2合成和VEGF的产生。
Objective To investigate the effect of the eukaryotic expression plasmid of specific small interfering RNA （siRNA）against COX-2 gene on the COX-2 expression and growth of human colon cancer HT-29 cells. Methods The COX-2 siRNA template DNA sequence for short hairpin RNA （shRNA） was designed and synthesized. The recombinant plasmid （pshCOX-2） was transfected into HT-29 cells. The effect of the recombinant plasmid on the COX-2 expression of human colon cancer HT-29 cells was detected by RTPCR and Western blot. Changes of the cell growth activity in response to transfected plasmid were evaluated by MTT assay and FCM. The methods of RIA and ELISA were respectively used to estimate the content of PGE2 and VEGF in supernatant. Results It was confirmed by restrictive enzyme digestion and sequence analysis that the recombinant plasmid was cloned and the aim sequence was obtained. The COX-2 expression of HT29 cells was inhibited at mRNA and protein levels 72 hours after transfected with the recombinant pshCOX-2. The growth of HT-29 cells was inhibited and the apoptotic ratio was significantly highly in COX-2 siRNA transfected group than those in control groups（P〈0. 05）. The content of PGE2 and VEGF in supernatant decreased significantly by transfecting pshCOX-2. Conclusion COX-2 siRNA expression plasmid pshCOX-2 successfully constructed can inhibit the expression of COX-2 gene,proliferation and synthesis of PGE2 and VEGF of HT-29 cells.
出处
《肿瘤防治研究》
CAS
CSCD
北大核心
2008年第10期705-710,共6页
Cancer Research on Prevention and Treatment
基金
江苏省135重点医学人才基金资助项目(RC2007076)
