人瘢痕疙瘩成纤维细胞Smad2特异siRNA的制备和活性鉴定

Preparation and characterization of Smad2 specific siRNA in human keloid fibroblast

目的:探讨应用小干扰RNA(small interfering RNA,siRNA)特异性抑制人瘢痕疙瘩成纤维细胞内Smad2 基因表达,筛选高效特异性siRNA。方法:根据siRNA设计原则,针对人Smad2 基因序列特征设计Smad2 特异siRNA(1 3),转染瘢痕疙瘩成纤维细胞,RT PCR检测siRNA对Smad2 基因的抑制效果。结果:Smad2 siRNA 3 可有效抑制瘢痕疙瘩成纤维细胞中Smad2 基因的表达。随siRNA 3 终浓度由50 nmol/L增加到100 nmol/L 及200 nmol/L,抑制效率逐渐增强(P<0.05);siRNA 3以终浓度200 nmol/L转染后24 h抑制效果最强,48 h 逐渐减弱,但仍明显抑制(P< 0. 05)。结论:应用RNA干扰技术可抑制瘢痕疙瘩成纤维细胞中Smad2 基因的表达,其抑制作用具有明显的时间、浓度依赖性。

Objective:To study the inhibition effect of Smad2 specific small interfering RNA(siRNA) on Smad2 gene expression in human keloid fibroblast,searching for the most efficient Smad2 specific siRNA.Methods: According to the principle of siRNA design,Smad2 specific siRNA(1-3) was designed and the keloid fibroblast were transfected.The level of Smad2 expressions were detected using semi-quantity RT-PCR. Results: Endogenous Smad2 expression were efficiently blocked in keloid fibroblast by Smad2 siRNA-3 in a dose- and time-dependent manner.Smad2 expression decreased significantly along with the increase of siRNA-3 dose within 50-200 nmol/L and was the lowest 24 h after transfection with 200 nmol/L siRNA-3. Conclusion: The expression of Smad2 in keloid fibroblast can be blocked efficiently in a dose- and time-dependent manner by specific siRNA.