基础医学与临床 ›› 2008, Vol. 28 ›› Issue (7): 707-712.

• 研究论文 • 上一篇    下一篇

D-siRNAs抑制A549细胞COX-2表达的研究

罗红 胡冬煦 陈平   

  1. 中南大学湘雅二医院心胸外科 中南大学湘雅二医院呼吸科 中南大学湘雅二医院呼吸内科
  • 收稿日期:2007-08-17 修回日期:2007-10-17 出版日期:2008-07-25 发布日期:2008-07-25
  • 通讯作者: 罗红

The expression of COX-2 in human pulmonary epithelial cells was suppressed by D-siRNAs

Hong LUO, Dong-xu HU, ping Chen   

  • Received:2007-08-17 Revised:2007-10-17 Online:2008-07-25 Published:2008-07-25
  • Contact: Hong LUO,

摘要: 目的 本研究拟利用长片段双链COX-2 RNA(dsRNAs)在体外经Dicer酶消化获得小RNA(D-siRNAs)抑制A549细胞COX-2的表达并降低PGE2的分泌,以望获得治疗气道炎症和肿瘤的一种新思路。方法 (1) IL-1β5ng/mL干预A549细胞0,3,6,9,12小时,Western blot法观察IL-1β诱导A549细胞COX-2蛋白表达的时间依赖性。(2) 用Trizol法抽提IL-1β刺激的体外培养的A549细胞总RNA,RT-PCR扩增COX-2基因。(3)设计两端带T7 promoter, SP6 promoter的COX-2 cDNA的PCR引物,通过PCR方法,获得两端带T7及SP6 promoter 的COX-2 DNA。(4) 用RiboMAX Large Scale RNA Production Systems-SP6 and T7 试剂盒体外将COX-2 DNA 转录为RNA。(5) 长片段COX-2 RNA 经Dicer酶消化为小RNA,纯化并获取小RNA(d-siRNAs)。(6) 用TransMessenger Transfection Reagent 将d-siRNAs转染A549细胞. (7)COX-2 mRNA表达用RT-PCR检测, 细胞培养上清液中PGE2含量用ELISA测定。结果 (1)IL-1β可刺激A549细胞COX-2蛋白质的表达,干预9小时后COX-2表达达到高峰(2)在体外,长片段COX-2 dsRNAs经Dicer酶消化可成功获得d-siRNAs(3) D-siRNAs可有效抑制A549细胞COX-2的表达并降低PGE2的分泌。结论 长片段双链COX-2 RNA(dsRNAs)在体外经Dicer酶消化获得小RNA(D-siRNAs)可有效抑制A549细胞COX-2的表达.

关键词: 环氧化酶2, A549细胞, D-SiRNAs, RNA干扰

Abstract: Objective Here we report the use of Dicer to cleave double-stranded RNA (dsRNA) into small interference RNAs (siRNAs) that can target multiple sites within an mRNA. Methods A549 cells were chosen to be incubated with 5ng/mL IL-1β for different time(0h,3h,6h,9h,12h,) to detect the time-dependent expression of COX-2.To generate the long dsRNA,the COX-2 gene (728bp) was amplified by PCR with a specific forward primer that contained a T7 promoter and a specific reverse primer that contained an SP6 promoter.Then,sense strand RNAs were generated by T7 RNA polymerase and antisense strand RNAs were generated by SP6 RNA polymerase.These single strand RNAs were annealed by the standard method. We mixed dsRNA with Dicer in reaction buffer. The mixture was incubated for 120 min at 37℃.We recovered siRNAs using RNA Purification Column.Transfections with diced siRNAs were performed using the TransMessenger Transfection Reagent in accordance with the manufacturer's instructions.COX-2 mRNA and protein were determined by RT-PCR and Western blot respectively. PGE2 was measured by ELISA. Results This study showed that IL-1β induced COX-2 protein expression in A549 cells.We recovered siRNAS that have been generated in vitro by Dicer. D-siRNAs significantly suppress the expression of COX-2 in human pulmonary epithelial .Conclusion In this study,we demonstrate that D-siRNAs significantly suppress the expression of COX-2 in human pulmonary epithelial

Key words: Cyclooxygense-2, A549 cell, D-siRNAs, RNAi