曹忻　邹云龙　徐红伟　杨具田

摘要：【目的】明確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Ⅲ VEGF 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。

关键词： 血管内皮生长因子（VEGF）；血管内皮生长因子受体；RNA干扰（RNAi）；颗粒细胞；绵羊

中图分类号： S826.92 文献标志码：A 文章编号：2095-1191（2017）02-0336-05

Abstract：【Objective】The paper studied effects of RNA interference（RNAi） on mRNA expression of vascular endothelial 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 designed for VEGF receptors Flt-1 and KDR/Flk-1 into ovine granule cells， and detected Flt-1 mRNA and KDR/Flk-1 mRNA 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 Ⅱ respectively， and the group imported with both Flt-1 interference 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 group Ⅰ， treatment group Ⅱ and treatment 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 Ⅱ dropped 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 group Ⅰ and treatment group Ⅲ could 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 group Ⅰ was significantly lower than that of control group since the 1st day. KDR/Flk-1 mRNA in both treatment group Ⅱ and 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.

Key words： vascular endothelial growth factor（VEGF）； vascular endothelial growth factor receptor； RNA interference（RNAi）； granule cell； ovine

0 引言

【研究意义】Senger等于1983年在肿瘤细胞分泌物中发现了一种促进内皮细胞增生、迁徙，抑制细胞凋亡，提高血管和微血管对大分子物质通透性的生长因子——血管内皮生长因子（Vascular endothelial growth factor，VEGF），目前，关于VEGF的研究多集中在人类医学，尤其是肿瘤方面（Espana-Serrano and Chougule，2016；Schicho et al.，2016；Wang et al.，2016）。在本课题组前期研究基础上推断VEGF对绵羊卵母细胞体外成熟和胚胎发育的促进作用可能与颗粒细胞有关，颗粒细胞可通过间隙连接为卵母细胞的成熟提供有利于其代谢的营养物质。因此，探究VEGF对绵羊颗粒细胞的影响，对后期研究VEGF在绵羊卵母细胞体外成熟和胚胎发育过程中的信号通路等具有重要意义。……