牛IFNτ受体特异介导胚胎源IFNτ体外调节子宫上皮中Wnt7a和Integrin ανβ3差异表达的研究

Induced expression of Wnt7a and Integrin ανβ3 in uterine by IFNτ secreted by Bovine blastocysts through its specific receptor-IFNAR is the key factor in regulating initiation of early embyro imlantation. Using different sources of embyros to induce the expression change of IFNAR in embryo or uterine epithelium cells and analyzing the source of IFNAR mediated IFNτ to regulate the expression of Wnt7a and Integrin ανβ3 in uterine epithelium cells can simulate the early implantation process in vivo and laid a good foundation for the interaction study of embyro and uterus during implantation and the embryo quality judgment. In this study, we take the expression level of IFNAR in in vivo bovine blastocysts as the control to analyze its expession in IVF and somatic cell cloned blastocysts by quantative RT-PCR, CONFOCAL and Western Blot. In addition, we use specific antagonist technique and gene knockout technique to investigate the source of IFNAR mediated IFNτ to regulate the expression of Wnt7a and Integrin ανβ3 in uterine epithelium cells. Finally, it should be an applicable reference for improving the pregnancy rate of those in-vitro produced ruminants’ embryos.

牛囊胚IFNτ通过特异受体IFNAR诱导子宫Wnt7a和Integrin ανβ3表达是调控、起始正常早期着床的关键因素。应用不同来源的胚胎体外诱导胚胎/子宫上皮细胞中IFNAR的表达，并进一步研究IFNτ-IFNAR调控子宫上皮细胞Wnt7a和Integrin ανβ3表达的分子机制，能够更真实地模拟体内早期着床过程，为体外研究胚胎与子宫在着床过程中的互作及据此准确判断胚胎质量奠定良好基础。因此，本研究拟采用定量PCR、CONFOCAL和Western blot等方法，以牛体内扩张囊胚中IFNAR基因的转录和翻译水平为对照，系统比较体外受精胚胎和体细胞克隆胚胎等体外囊胚中IFNAR基因的表达变化。同时，借助特异拮抗技术与基因敲除技术，系统探究IFNτ-IFNAR体外诱导子宫上皮细胞Wnt7a和Integrin ανβ3蛋白差异表达的调控机制，为进一步提高牛体外胚胎的移植妊娠率提供参考。

牛囊胚分泌的IFNτ通过特异受体IFNAR，对其子宫中Wnt7a和Integrin ανβ3蛋白的诱导是调控、起始其正常早期着床的关键因素，但是，目前对这类反刍动物胚胎与子宫互作调控的具体机制尚不清楚。本研究采用定量PCR、CONFOCAL、Western检测、IFNAR特异拮抗和基因条件性敲除等方法，同时结合牛体外胚胎与其子宫上皮细胞体外共培养体系，以牛体内扩张囊胚中IFNAR基因在转录和翻译水平上的变化为对照，系统比较经IVF（体外受精）、PA（孤雌激活）和体细胞克隆（SCNT）等体外囊胚中IFNAR基因的表达变化；同时结合胚胎-子宫上皮体外共培养体系，系统探究了牛胚胎源的IFNτ，究竟通过何种IFNAR调控方式，体外诱导其子宫上皮细胞发生Wnt7a和Integrin ανβ3蛋白的差异表达，并发现：IFNτ主要在这四类牛囊胚的滋养层细胞表达，且IFNAR的两型受体-IFNAR1与IFNAR2也主要在其囊胚滋养层细胞表达，而IFNAR1的表达与胚胎源的IFNτ表达密切相关。利用牛囊胚-子宫上皮细胞进行体外共培养发现：子宫上皮细胞的IFNAR1和IFNAR2出现上调，而Integrin αv β3, WNT7A, 与ISG15却出现下调。特异性拮抗其胚胎源IFNAR1和IFNAR2后，子宫上皮中Integrin αv β3, WNT7A, 与ISG15都显著下降，利用CRISPER-cas9技术敲降其胚胎源IFNAR1和IFNAR2后，子宫上皮也出现同样的下调变化，但是敲降IFNAR2的作用明显优于敲降IFNAR1。对体外培养的牛子宫上皮细胞分别进行IFNAR1和IFNAR2基因敲降后，再与上述四类牛囊胚进行体外共培养后还发现：与IVF胚胎共培养，其Integrin αv β3的表达被显著抑制，而WNT7A 的表达则不受抑制。与PA胚胎共培养，其Integrin αv β3和WNT7A 的表达也都不受抑制。由此可见：牛胚胎源的IFNτ主要通过其自身的IFNAR1以及子宫上皮细胞的IFNAR2特异性调控其胚胎早期着床的子宫容受态。