RNA干扰沉默酸性鞘磷脂酶1基因对人成纤维细胞凋亡的影响

目的为研发保护女性生殖细胞的小分子干扰RNA（siRNA）类药物，以人包皮成纤维细胞（HFF）为模型，检测靶向凋亡关键基因酸性鞘磷脂酶1（SMPD1）基因的siRNA对HFF凋亡的抑制作用。方法设计合成三对靶向SMPDI基因的siRNA并体外转染HFF细胞，转染后48h用丝裂霉素（MMC）诱导细胞凋亡。荧光定量逆转录-聚合酶链反应（O-RT-PCR）法及免疫印迹（Western blot）检测siRNA转染后SMPD1在mRNA水平和蛋白水平的变化，Annexin V—PI双染检测细胞凋亡率。用脂质体（lipofectamine 2000）和无干扰siRNA（NT siRNA）作为对照。结果siRNA1、2、3对SMPD1基因mRNA的抑制效率分别为（67．0±9．0）％、0％、（45．0±2．1）％，以siRNA1最高，蛋白水平的抑制也呈现相同变化。siR-NA1组细胞凋亡率为15．2％，明显低于MMC组（26．3％）。结论靶向SMPD1的siRNA可以抑制人包皮成纤维细胞凋亡。

The effect of RNA interference targeted on SMPD1 on apoptosis in HFF cells

Objective： To study the anti-apoptotic effect gene in human foreskin fibroblast （HFF） cells. of small interfering RNA （siRNA） targeted on SMPD1 Methods： We designed and synthesized 3 small interference RNA （siRNA） sequences targeted on SMPD1 and transfected them into HFF cells in vitro. Forty eight hours after siRNA transfection, HFF cells were treated with mitomycin C （MMC） to induce apoptosis. SMPD1 mRNA was measured with quantitative RT-PCR and protein by Western blot. Apoptotic rate of the cells was determined by flow cytometer （FCM） after AnnexinV/PI staining. Cells in control group were treated with mock transfection （lipofectamine 2000 only） or negative control siRNA. Results. To SMPD1 gene expression, the inhibitory rates by siRNA1,2,3 were （67.0 ± 9.0）%, 0% and （45. 0 ＋ 2. 1）%, respectively. The SMPD1 protein decreased significantly in siRNA1 group.The apoptotic rate of the cells in siRNA1 group was 15.2%, which was significantly lower than that in control group （26.3%）. Conclusion： siRNA targeted on SMPD1 can protect HFF cells from apoptosis.