目的 设计、合成及筛选光老化相关基因—转化生长因子-β1(transforming growth factor-β1,TGF-β1)小分子干扰RNA (siRNA),了解基因调控效果,用以探索皮肤抗衰老基因治疗可能的新途径.方法 应用RNA设计软件,设计并合成针对小鼠TGF-β1 mRNA的3对21核苷酸siRNA,转染小鼠成纤维细胞,检测转染效率及细胞活性;分别在基因和蛋白水平检测TGF-β1表达.结果 TGF-β1基因siRNA工作浓度为150nmol/L,Lipofectamine2000脂质体4μL/孔(24孔板)时转染率最高(84.0％).综合考虑转染率与细胞活性,选用0.5μL/孔(转染率为72.4％).与对照组相比,转染TGF-β1 siRNA的小鼠皮肤成纤维细胞TGF-β1表达下调,其差异有统计学意义(P＜0.05),以siRNA-S1最显著,基因水平抑制率达96.9％,蛋白水平抑制率达62.0％.结论:TGF-β1 siRNA转染小鼠成纤维细胞最佳条件是Li-pofectamine2000脂质体0.SμL/孔,siRNA-S1有效抑制TGF-β1蛋白表达时间可达72h.%Objective To design,synthesize and screen small interfering RNA (siRNA) targeting at transforming growth factor-β1 TGF-β1) and to understand the effect of gene regulation so as to explore a possible way for the gene therapy of skin anti-aging.Methods 3 pairs of 21 nucleotide siRNAs directed to mouse TGFβ1 mRNA were designed and synthesized by utilizing RNA design software;these siRNAs were respectively transfected into fibroblastsby oligofectamine package;transfection efficiency and cell activity were detected;the expression of TGFβ1 was detected in gene and protein level.Results When the work concentration of siRNA was 150nmol/L and that of Lipofectamine2000 was 4μL / well (24-well plates),transfection efficiency of fibroblasts was highest (84.0％);considering cell viability and transfection efficiency,0.5μL/well (72.4％)was chosen;compared with bland controls,the expression of TGFβ1 protein level was markedly down-regulated in siRNA transfected fibroblasts and the difference was of statistical significance (P ＜ 0.05);the inhibitory rate of the siRNA of the target-1 was highest up to 96.9％ in mRNA level and 62.0％ in protein level.Conclusions The optimal condition of TGF-[β1 siRNA transfecting fibroblasts was Lipofectamine2000 0.5μL/well;siRNA-S1 can effectively inhabit the protein expression of TGF-β 1 for 72 hours.
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