短发夹 RNA 介导 RNA 干扰的时间 和剂量效应研究

用 RNA 干扰 (RNA interference , RNAi) 技术抑制哺乳动物细胞中外源报告基因的表达,以探讨该过程中 RNAi 作用的剂量和时间效应 . 应用 Lipofectamine 2000 将外源报告基因的表达载体与编码短发夹 RNA (short hairpin RNA , shRNA) 的质粒共转染 HEK293H 细胞,观察 shRNA 载体对报告基因的抑制效应 . 转染后, shRNAs 的瞬时表达可特异地抑制细胞内报告基因的表达 . 在共转染后 12 , 24 , 48 , 60 , 72 , 96 h 时检测 EGFP (enhanced green fluorescent protein , EGFP) 基因 mRNA 及蛋白质表达水平,结果显示, EGFP mRNA 及蛋白质表达在 12 h 时略有降低, 24~48 h 时表达逐渐降低, 48~72 h 时降低最明显,其后 EGFP 表达水平逐渐恢复 . 提示该过程中 RNAi 效应呈现由弱到强、又由强到弱的逐渐消逝趋势 . 共转染一系列剂量比例的 EGFP 干扰载体与靶载体的结果表明,在一定剂量范围内, RNA 干扰载体所介导的抑制效应与干扰载体剂量大小有关,当其剂量进一步加大足以抑制外源基因表达时,抑制效应则维持在一“平台期” . 此外,通过 RNAi 抑制 HeLa 细胞、 HEK293 细胞中荧光素酶基因的表达, 荧光素酶活性变化也表现出上述类似的效应 . 这些结果表明,在体外哺乳动物细胞中,基于表达载体的 RNAi 作用呈现剂量和时间依赖性效应 . 这为基于载体表达的 RNAi 技术应用研究提供了一定的理论参考及依据 .

Time and Dose Effect of RNA Interference Mediated by Short Hairpin RNA

To investigate whether there exits the dose- and time- dependent effect of RNA interference(RNAi) when the introduced extraneous reporter gene was suppressed by RNAi in mammalian cell lines.The expression vectors carrying reporter component were cotransfected with the plasmids coding short hairpin RNAs(shRNAs) into HEK293H cell using lipofectamine 2000 reagent, and the consequent inhibitory effect mediated by RNA interference was observed. After transfection, the transient expression of shRNAs could specifically inhibit the extraneous reporter in mammalian cell.The expression of mRNA and protein of enhanced green fluorescent protein(EGFP)gene was determined at 12, 24, 48, 60, 72, 96 h after transfection in HEK293 cell. The results showed that the decrease of EGFP mRNA or protein level was not obvious at 12 h, but gradually became more evident during from 24 to 48 h.The decrease achieved the maximal degree during from 48 to 72 h, then became weakened and restored subsequently. It indicated that the effect of RNA interference underwent a tendency of from weak to strong, then from strong to weak, and ultimately disappeared gradually. The efficiency of inhibition caused by RNAi was related to the dose of RNA interfering vector within a confined limit in HEK293H cell cotransfected with a series of dose-proportional vectors as pd1EGFP and psh-d1EGFP, whereas it nearly kept constant when the dose of interfering plasmid was sufficient to depress the expression of extraneous genes. Simultaneously, the variation of luciferase activity also displayed the similar effect when its expression was suppressed by RNAi in HEK293H or HeLa cell.. It concluded that vector-based RNA interference took on time- and dose- dependent effect in mammalian cell, which provides certain theoretical reference or valuable clue to the utility of RNAi.