利用颗粒细胞筛选绵羊VEGFR有效干扰片段的研究

The research of screening the effective RNA interference fragments of vascular endothelial growth factor receptors of ovine with granular cells

为了进一步探究血管内皮生长因子(VEGF)对绵羊卵母细胞成熟和胚胎发育的作用,试验采用电穿孔技术(Electroporation)将针对血管内皮生长因子受体(VEGFR)中的两个受体FLT1和KDR/FLK-1设计的6条双链小RNA导入到绵羊颗粒细胞内,通过实时定量PCR检测FLT1和KDR/FLK-1表达量的变化,筛选出针对VEGFR的有效干扰片段。结果表明:试验组F2(FLT1-siRNA-2)第1~3天的FLT1 mRNA相对表达量分别为7.17×10^(-6)±4.97×10^(-8)、5.92×10^(-6)±4.73×10^(-8)和1.14×10^(-5)±2.84×10^(-7),极显著低于其他FLT1试验组(P<0.01),且FLT1 mRNA相对表达量相差100倍或10倍;试验组K1(KDR/FLK-1-siRNA-1)第1~3天KDR/FLK-1 mRNA相对表达量分别为1.46×10^(-7)±6.08×10^(-9)、2.80×10^(-7)±1.82×10^(-8)和7.99×10^(-7)±2.60×10^(-8),极显著低于其他KDR/FLK-1试验组(P<0.01),且KDR/FLK-1 mRNA相对表达量相差100倍或10倍。从第4天开始试验组F2 FLT1 mRNA相对表达量和试验组K1 KDR/FLK-1 mRNA相对表达量逐渐增加,到达第9天与其他组趋于相近。最终确定针对FLT1和KDR/FLK-1最有效的干扰片段分别是FLT1-siRNA-2和KDR/FLK-1-siRNA-1。

For further research of vascular endothelial growth factor （VEGF） on ovine oocytes maturation and embryonic development. Electro- potation was used to import 6 double chain of small RNA interference （RNAi） fragments which designed for VEGF receptors FLT1 and KDR/ FLK - 1 into ovine granule cells. Real - time quantitative PCR were used to detect the changes of FLT1 mRNA and KDR/FLK - 1 mRNA levels of granule cells,and to screen the most effective fragments aimed to the two receptors of VEGF. The results showed that FLT1 mRNA levels from 1st to 3rd day in F2 experimental group were 7.17 × 10 ^-8 ±4.97 × 10^ -8 ,5.92 × 10 ^-6 ±4.73 × 10 ^-8 and 1.14 × 10^ -5 ± 2.84 × 10 ^-7 ,which significandy lower than the other FLT1 interference groups （P 〈0.01 ） ,and the differences of FLT1 mRNA relative expressions were 100 times or 10 times; KDR/FLK - 1 mRNA levels from 1st to 3rd day in K2 group were 1.46 × 10 ^-7 ±6.08 × 10 ^-9,2.80 × 10^ -7 ± 1.82 × 10 ^-8 and 7.99 × 10 ^-7 ±2.60 × 10^ -8 ,which significantly lower than other KDR/FLK - 1 interference groups （P 〈0.01 ） ,and the differences of KDR/ FLK - 1 mRNA relative expressions were 100 times or 10 times. FLT1 mRNA level in F2 group and KDR/FLK - 1 mRNA level in K1 group were both gradually increased since the 4th day, that displayed similar tend to other groups on the 9th day. In this research,FLT1 - siRNA - 2 fragment and KDR/FLK - 1 - siRNA - 1 fragment were confirmed as the most effective interference fragments for FLT1 mRNA and KDR/FLK - 1 mRNA,respectively.