siRNA真核表达载体对大鼠耳蜗前体细胞Notch3基因表达的影响

Blocking effect of vector-based siRNA on Notch3 gene expression in rat cochlear progenitor cells

目的构建Notch3基因的siRNA真核表达载体,探讨其对大鼠耳蜗前体细胞中Notch3基因表达的影响。方法机械分离并胰酶消化新生大鼠耳蜗感觉上皮,悬浮培养基培养耳蜗前体细胞。根据Notch3基因序列设计并合成2对siRNA,插入pSilencer4.1-CMV neo载体,进行酶切及测序鉴定。采用脂质体法向大鼠耳蜗前体细胞转染重组质粒,采用Real time-PCR和Western blotting法检测重组质粒对Notch3基因的干扰效果。结果经前体细胞标记物Abcg2染色鉴定证实原代培养的细胞球为耳蜗前体细胞。酶切及测序鉴定证实成功构建了siRNA真核表达载体pSilencer-Notch3-S1和pSilencer-Notch3-S2。Real-time PCR和Western blotting检测结果显示所构建的Notch3基因真核表达载体成功地干扰了目的基因的表达。与空白对照组比较,转染pSilencer-Notch3-S1和pSilencer-Notch3-S2的细胞Notch 3 mRNA和蛋白表达量均有明显减少,而转染空质粒的细胞与空白对照组比较无显著差异。结论成功构建了大鼠Notch3基因的RNAi真核表达载体,该载体在大鼠耳蜗前体细胞中对Notch3基因的表达可发挥抑制作用。

Objective To explore the blocking effect of siRNA on the expression of Notch3 gene in rat cochlear progenitor cells using siRNA eukaryotic expression vector. Methods The sensory epithelial cells of neonatal rat cochlea were mechanically isolated, and then they underwent trypsin digestion and suspension culture to acquire cochlear progenitor cells. Immunfluorescence staining of Abcg2 expression was applied to confirm the acquired cochlear progenitor cells. Two siRNA cDNAs were synthesized according to the Notch3 gene sequence and cloned into the vector pSilencer4.1-CMV neo, namely pSilencer-Notch3-S1 and pSilencer-Notch3-S2 respectively, and they were further identified by restriction endonuclease digestion analysis and DNA sequencing. The cochlear progenitor cells were then transfected with pSilencer-Notch3-S1 and pSilencer-Notch3-S2. The interfering effect was detected by Real-time PCR and Western blotting. Results The clonal growth cell spheres were acquired and identified to contain Abcg2 positive cochlear progenitor cells. The vectors were successfully constructed and confirmed by DNA sequencing restriction endonuclease digestion analysis. DNA sequencing showed that the two target segments were cloned into pSilencer4.1- CMV neo-vector respectively. Western blotting showed that the protein expression decreased significantly in pSilencer-Notch3-S1 transfected cochlear progenitor cells. Real-time PCR demonstrated a significant decrease in expression of Notch3 mRNA in both pSilencer-Notch3-S1 group and pSilencer-Notch3-S2 group. Conclusion The vector-based siRNA on Notch3 gene can effectively inhibit the expression of Notch3 gene in rat cochlear progenitor cells.