目的 观察过表达与沉默DNA结合抑制因子2(ID2)基因对人主动脉平滑肌细胞(HA-VSMC)表型转化的影响.方法 设计合成靶向ID2的小干扰RNA (siRNA)并转染HA-VSMC,实时荧光定量聚合酶链反应(FQ-PCR)筛选出沉默效率最高的siRNA序列.生物合成人ID2基因序列,将其插入pIRES2-ZsGreen1质粒载体,构建过表达ID2重组质粒载体pIRES2-ZsGreen1-ID2并鉴定.HA-VSMC分5组培养:空白对照组、空质粒对照组、ID2过表达组、siRNA对照组、ID2 siRNA组.FQ-PCR及Western blot检测各组ID2、平滑肌22α(SM22α)、α.平滑肌肌动蛋白(α-SMA)、骨桥蛋白(OPN)mRNA及蛋白表达.结果 设计并合成有较高沉默活性的靶向ID2的siRNA,并成功构建了pIRES2-ZsGreen1-ID2过表达质粒.与空白对照组、空质粒对照组、siRNA对照组比较;ID2过表达组的ID2、OPN mRNA和蛋白表达均显著升高[ID2 mRNA:2.637±0.053;OPN mRNA:1.692±0.086;ID2:0.580±0.017;OPN:0.460±0.015],SM22α、α-SMA mRNA和蛋白表达均显著降低[SM22α mRNA:0.367±0.048;α-SMA mRNA:0.428±0.031;SM22α:0.100±0.004;α-SMA:0.063±0.010] (P<0.01);而ID2 siRNA组ID2、OPN mRNA和蛋白表达均显著降低[ID2 mRNA:0.322±0.046;OPN mRNA:0.250±0.056;ID2:0.061±0.007;OPN:0.066±0.006],SM22α、α-SMA mRNA和蛋白表达均显著升高[SM22α mRNA:1.827±0.041;α-SMA mRNA:1.653±0.088;SM22α:0.547±0.009;α-SMA:0.671±0.051,P<0.01].结论 过表达ID2基因可诱导HA-VSMC发生由收缩型向合成型的表型转化,而沉默该基因可抑制这一过程.
Objective To study the effect of DNA binding inhibitory factor 2 (ID2) overexpression and silence on human aortic vascular smooth muscle cells (HA-VSMC) phenotype switch.Methods The small interfering RNA (siRNA) targeting ID2 was designed and synthesized,and transfected into HA-VSMC.Quantitative real-time polymerase chain reaction (FQ-PCR) was used to select a siRNA which had the most efficient silence ability.The ID2 gene sequences were biosynthesized,inserted into pIRES2-ZsGreen1 plasmid vector,and the ID2 recombinant overexpression plasmid vector pIRES2-Zs-Green1-ID2 were constructed and identified.HA-VSMC were divided into five groups:blank control group,empty plasmid control group,ID2 overexpression group,siRNA control group,ID2 siRNA group.FQ-PCR and Western blotting assays were used to detect the expression of ID2,smooth muscle 22α(SM22α),α-smooth muscle actin (α-SMA) and osteopontin (OPN),respectively.Results The siRNA targeting ID2 with higher silence ability was successfully designed and synthesized,and the overexpression plasmid pIRES2-ZsGreen1-ID2 constructed.The ID2 and OPN mRNA and protein expression levels in ID2 overexpression group were significantly higher than in blank control group,empty plasmid control group and siRNA control group [ID2 mRNA:2.637 ± 0.053;OPN mRNA:1.692 ± 0.086;ID2:0.580 ±0.017;OPN:0.460 ±0.015],SM22,and α-SMA mRNA and protein expression levels were significantly lower [SM22α mRNA:0.367 ± 0.048;α-SMA mRNA:0.428 ± 0.031;SM22α:0.100 ±0.004;α-SMA:0.063 ±0.010] (P <0.01),and inID2 siRNA group,ID2 and OPN mRNA and protein expression levels were significantly lower [ID2 mRNA:0.322 ± 0.046;OPN mRNA:0.250 ± 0.056;ID2:0.061 ±0.007;OPN:0.066 ±0.006],SM22,and α-SMA mRNA and protein expression levels were significantly higher [SM22α mRNA:1.827 ± 0.041;α-SMA mRNA:1.653 ± 0.088;SM22α:0.547±0.009;α-SMA:0.671±0.051] (P<0.01).Conclusion OverexpressionofID2genecaninduce HA-VSMC to switch from a contractile to synthetic phenotype,while silencinh the gene can inhibit this process.