猪孤雌胎儿发育阻滞的影响因素研究

The mechanism of mammalian parthenogenesis is of great interest, yet not totally elucidated. As the physiological structure is similar to human,porcine model has become a hot tool for medicine and production studies.Pig parthenogenesis embryo can develop through early preimplantation stages,but growth retarded at embryonic day 28,Our laboratory has found that the parthenote fetus and placenta grew slower than those of controls and disruption expression of imprinted genes in porcine parthenogenesis fetuses.And so far,little has been known the mechanism of porcine parthenogenesis retarded development.In order to further investigate the molecular mechanism of parthenogenesis development retard in porcine reproductive biology,we examined the imprinting status of candidate genes in parthenote fetus and placenta by real-time q-PCR,and determined whether such expression of imprinted genes is regulated by DMR of methylation using nMS-PCR.We intend to identify the gene expression profile and the pathway analysis of parthenogenesis fetuses and placenta using Porcine GeneChip Microarrays,including the expression of imprinted gene,DNA Methylation,chromosome remodeling,histone acetylation and methylation,DNMT and RNA edition.The purpose of this study is to provide strong evidence to facilitate our understanding of the molecular mechanism of porcine parthenogenesis retarded development,and the possibility of birthing the full-term development parthenote pig.

哺乳动物孤雌生殖机制是备受关注但又了解甚少的科学问题。近年来猪因其与人类相似的生理结构已成为医学模型和生殖领域的研究热点。已有文献和本课题组前期实验表明，猪孤雌胚胎在体内可着床并发育至28天，但猪孤雌胎儿发育延缓且印迹基因发生重建，上述现象的产生机制尚不明确。为进一步揭示猪孤雌胎儿发育阻滞的相关分子机制，本研究拟以猪孤雌发育胎儿、胎盘为研究对象，采用实时荧光定量PCR比较分析胎儿及胎盘的印迹基因表达差异，进一步通过巢式甲基化特异性PCR(nMS-PCR)对印迹基因的差异甲基化区（DMR）进行测定，探索印迹基因表达情况及其甲基化模式；采用猪基因组表达芯片对胎儿及胎盘组织进行基因表达差异分析，对染色体重塑、DNA甲基化、组蛋白的甲基化及乙酰化、DNMT、RNA编辑等通路及相关基因表达差异进行分析，旨在阐明猪孤雌胎儿发育阻滞的分子机制，为构建发育到期的孤雌生殖猪奠定理论基础。

哺乳动物孤雌生殖机制是备受关注但又了解甚少的科学问题。本课题结果表明，猪孤雌胚胎在体内可着床并发育，但其发育延缓且印迹基因紊乱表达，尤其是印迹基因H19/Igf2 的异常表达，可能是哺乳动物孤雌发育阻滞的重要因素。主要的学术成果包括：1. 验证了印记基因H19/Igf2,NNAT, MEST等基因的基因表达模式； 2. 提出印记基因的紊乱表达可能是孤雌发育阻滞的主要因素； 3. 首次提出孤雌胎盘发育阻滞的因素为VEGF120的过表达导致； 4. 验证了再克隆方法可延长孤雌猪体内发育时间至 39天；5. 筛选出睾丸酮可对H19/Igf2 的基因表达模式进行调控。 此外，通过该项目的实施，本发表相关SCI 论文11篇。.本项目的顺利实施，为后续研究利用猪孤雌细胞为研究对象，采用睾丸酮处理，对异常表达的H19/Igf2 进行改善；进一步通过Crispr Cas9对紊乱的印迹基因进行表达调控，使孤雌细胞中的基因组印迹趋于正常化；利用体细胞核移植（SCNT）、胚胎移植技术、B-超检测、性别鉴定、Southern blot、q-PCR以及RNA-seq等实验手段，探索延长孤雌猪体内发育时间或构建发育到期孤雌猪的可能性为构建发育到期的孤雌生殖猪奠定理论及实验基础。