下调DKK2表达对小鼠子宫内膜基质细胞增殖和凋亡的影响

[目的] 本试验旨在研究RNA干扰Dickkopf-2基因（DKK2）表达对小鼠子宫内膜基质细胞增殖和凋亡的影响。[方法] 设计并合成3条针对DKK2基因的siRNA，在转染试剂LipofectamineTM 2000介导下转染小鼠子宫内膜基质细胞，用RT-qPCR法检测转染前、后DKK2基因的表达量，筛选干扰效率最高的1条siRNA。用该siRNA转染小鼠子宫内膜基质细胞24 h，利用Caspase3活性和Annexin-V FITC/PI双染法检测干扰DKK2基因对细胞凋亡的影响；用CCK8细胞计数法检测该siRNA干扰DKK2基因不同时间对小鼠子宫内膜基质细胞增殖的影响。[结果] 3条siRNA均能有效抑制DKK2基因的表达，RT-qPCR结果显示DKK2基因表达水平显著降低（siRNA2，P<0.05；siRNA1、siRNA3，P<0.01）；CCK8法结果显示siRNA干扰DKK2后对小鼠子宫内膜基质细胞有明显的抑制作用（24 h、48 h，P<0.05）；Caspase3活性检测和Annexin-V FITC/PI双染法结果显示，siRNA1转染小鼠子宫内膜基质细胞24 h后，Caspase3活性明显增加（P<0.01），细胞凋亡率明显增加（P<0.01），凋亡率为18.48%。[结论] 利用RNA干扰技术沉默DKK2基因的表达可以明显抑制小鼠子宫内膜基质细胞的增殖并促进凋亡。
[Objectives] This study is aimed to investigate the effects of silencing of DKK2 gene expression by siRNA on the proliferation and apoptosis of mouse endometrial stromal cells. [Methods] We professionally devised and synthesized three siRNAs against DKK2,then transfected them into endometrial stromal cells by lipofectamineTM 2000 assay. The silencing rate of predesigned siRNAs that targeted DKK2 was detected by real-time quantitative RCR(RT-qPCR) analysis and the most efficient siRNA was screened out. Furthermore,the best silencing siRNA that targeted DKK2 was transfected into endometrial stromal cells,and cell proliferation inhibition rates and Caspase3 activity were determined by CKK8 and Caspase3 assay. The effect of siRNA on cell apoptosis was determined by flow cytometry. [Results] The three siRNAs were successfully transfected into mouse endometrial stromal cells and the cells were green under fluorescence microscope. The experiment of RT-qPCR showed that the expression of DKK2 mRNA in treatment groups was lower than control groups. The best sequence of siRNA for silecing of DKK2 gene expression was siRNA1 with the silencing rate of up to 60% 24 h after transfection. The stromal cells proliferation was significantly inhibited at the time points of 24 h,48 h after transfection. The Caspase3 activity of stromal cells was significantly increased in siRNA-DKK2 transfection group(P<0.01). Meanwhile,the apoptosis rate of stromal cells was significantly increased in siRNA-DKK2 transfection group,and the rate was 18.48%(P<0.01). [Conclusions] Application of RNA interference against DKK2 gene on mouse endometrial stromal cells can inhibit the proliferation and promote apoptosis