RNAi对绵羊颗粒细胞体外培养过程VEGF及其受体mRNA表达的影响

Effects of RNAi on m RNA expression of VEGF and VEGF receptor during granule cell in vitro culture of ovine

【目的】明确RNA干扰(RNAi)对绵羊颗粒细胞体外培养过程中血管内皮生长因子(Vascular endothelial growth factor,VEGF)及其受体mRNA表达的影响,为开展VEGF在绵羊卵母细胞体外成熟和胚胎发育过程中信号通路的相关研究打下基础。【方法】采用电穿孔技术将针对VEGF两个受体Flt-1和KDR/Flk-1的两条有效双链小RNA导入绵羊颗粒细胞内,利用实时荧光定量PCR检测体外培养绵羊颗粒细胞各培养时间VEGF、Flt-1和KDR/Flk-1 mRNA表达水平的变化。设定筛选出的Flt-1和KDR/Flk-1有效干扰片段分别为试验组Ⅰ和试验组Ⅱ,两个干扰片段共同作用为试验组Ⅲ,无效的干扰片段为对照组。【结果】绵羊颗粒细胞体外培养过程中,VEGF mRNA在各时间段的相对表达量是Flt-1 mRNA相对表达量的102倍、KDR/Flk-1 mRNA相对表达量的103倍;培养第1 d时,试验组Ⅰ、试验组Ⅱ和试验组ⅢVEGF mRNA相对表达量分别为2.62×10-2、3.54×10-2和2.94×10-2,均极显著高于对照组(P<0.01,下同),3个试验组的VEGF mRNA表达量从第2 d开始降低,直到第6 d与对照组趋于一致。试验组ⅡFlt-1 mRNA相对表达量从培养第1 d的1.02×10-1逐渐降至第7 d的8.99×10-4,且一直极显著高于其他组;试验组Ⅰ和试验组Ⅲ在前3 d几乎无Flt-1 mRNA表达,随着培养时间的延长逐渐呈上升趋势,第8 d时各组趋于一致。试验组Ⅰ的KDR/Flk-1 mRNA相对表达量从第1 d开始极显著低于对照组;试验组Ⅱ和试验组Ⅲ在前3 d几乎无KDR/Flk-1 mRNA相对表达,随着培养时间的延长呈上升趋势,第9 d时各组趋于一致。【结论】两个有效干扰片段在绵羊颗粒细胞体外培养过程中起到很好的干扰作用,可改变VEGF、Flt-1及KDR mRNA的表达量,进而影响颗粒细胞相关生长信号的传输。

[Objective]The paper studied effects of RNA interference（RNAi） on mRNA expression of vascular en-dothelial growth factor（VEGF） and its receptors in the process of granule cells cultured in vitro, in order to lay a foundation for research in signaling pathway of VEGF during process of oocyte in vitro maturation and embryonic development.[Method]This research used electroporation to import two double chain small interfering RNA fragments which were de-signed for VEGF receptors Flt-1 and KDR/Flk-1 into ovine granule cells, and detected Flt-1 mRNA and KDR/Flk-1 mR-NA levels variation of ovine granule cells cultured in vitro at different times by real-time quantitative PCR. The researchers named the groups imported with Flt-1 interference fragment and KDR/Flk-1 interference fragment which were screened out in early experiment as treatment group Ⅰand treatment group Ⅱrespectively, and the group imported with both Flt-1 in-terference fragment and KDR/Flk-1 interference fragment as treatment group Ⅲ, and the group imported with interference fragment which had no RNAi effect as control group. [Result]Results indicated that, during the process of ovine granule cells cultured in vitro, mRNA expression level of VEGF was 102 times higher than that of Flt-1 and 103 times higher than that of KDR/Flk-1. On the 1st day, the VEGF mRNA expression levels of treatment groupⅠ, treatment groupⅡand treat-ment group Ⅲ were 2.62×10-2, 3.54×10-2 and 2.94×10-2 respectively, which were significantly higher than that of control group（P〈0.01, the same below）. The expression levels of the three treatment groups reduced since the 2nd day and tended to be similar to that of control group on the 6th day. During the culture process, Flt-1 mRNA level of treatment group Ⅱdropped from 1.02×10-1（on the 1st day） to 8.99×10-4（on the 7th day）, and it had been significantly higher than that of other groups. Flt-1 mRNA expressions of both treatment groupⅠand treatment groupⅢcould be barely detected, then, as time passing by, they rose and tended to be similar to those of other groups on the 8th day. KDR/Flk-1 mRNA level of treatment groupⅠwas significantly lower than that of control group since the 1st day. KDR/Flk-1 mRNA in both treatment group Ⅱand treatment group Ⅲ could be barely detected from the 1st day to the 3rd day, and they rose with time passing by and tended to be similar to those of other groups on the 9th day. [Conclusion]Two pairs of effective interference fragments have good performance on interfering during the process of ovine granule cells in vitro culture, which results in change of mRNA expression levels of VEGF, Flt-1 and KDR mRNA, and affects transmission of growth signal concerning granule cells.