p66shc-ROS轴介导猪早期胚胎体外发育阻滞机理的研究

P66shc (66-kilodahon isoform of Shc gene products) is one of the three ShcA protein family member with similar molecular weight, which is encoded by SHC oncogene of mammal. It has been demonstrated that p66shc protein plays an important role in cell apoptosis caused by reactive oxygen species (ROS); it has also been found that ROS in embryos is one of the most important reasons leading to embryo development block in vitro, but the mechanism is not clear yet. Therefore, this research focuses on p66shc as the research target gene, which is closely related to ROS reaction leading to embryonic development block. In order to prove this hypothesis, the experiments are carried out as following:(1)the levels of ROS and p66shc expression are analyzed between development block and normal development embryos in order to figure out the relation betwwen p66shc gene and ROS level for describing the expression pattern of p66shc-ROS-embryo development;(2)the embryonic ROS level and developing efficiency are researched through microinjecting of p66Shc interference fragment into oocytes or embryos using RNAi technology in vitro; (3)the embryonic ROS level and developing efficiency are researched through adding antioxidants or p66Shc antibody in the culture medium in vitro. In conclusion, the research focus on porcine embryo p66shc-ROS-embryo development shaft in order to find how to control the mechanism of embryo developing block by the p66shc gene, trying to elucidate the regulation mechanism of p66shc on porcine embryo development block in order to further improve the efficiency of porcine early embryos development in vitro.

p66shc是原癌基因SHC编码三个分子量相近的ShcA蛋白质家族中成员之一。研究发现p66shc在由活性氧(reactive oxygen species, ROS)引起细胞凋亡中发挥重要的作用；而在胚胎早期发育过程中，胚胎内ROS是导致胚胎体外发育阻滞的重要原因之一，但是其具体调控机制不详。为此，本研究选择p66shc基因作为引起猪胚胎发育阻滞的靶基因，首先研究发育正常与阻滞的猪胚胎内该基因和ROS水平的关联性，构建p66shc-ROS-胚胎发育的动态模式；然后通过上调（在高氧浓度下等方法）或下调该基因的表达（显微注射RNAi片段，添加抗氧化剂和抗体等）等方法，验证该基因对猪胚胎内ROS水平及发育效率的调控作用。总之，该研究着眼于探索猪胚胎p66shc-ROS-胚胎作用轴调控胚胎发育阻滞的机制，为提高猪胚胎体外发育效率奠定基础。

已有研究发现，P66Shc在由活性氧(reactive oxygen species, ROS)引起细胞凋亡中发挥重要的作用；而在胚胎早期发育过程中，胚胎内ROS是导致胚胎体外发育阻滞的重要原因之一，但是其具体调控机制不详。为此，选择P66Shc基因作为由于ROS引起猪胚胎发育效率低下的靶基因进行研究。首先分离培养成纤维细胞，分别比较其细胞的生长情况（生长曲线和倍增时间等），研究其细胞内ROS水平与P66Shc的联系，其实验研究结果表明P66Shc基因的表达与氧浓度存在正相关的作用，为后续影响胚胎发育的研究奠定理论基础。接着在构建了猪卵母细胞体外成熟培养技术和早期胚胎体外培养技术体系，发现了通过选择II型极体的猪卵母细胞进行后续研究可提高胚胎体外发育效率。在此基础上，通过研究猪早期胚胎在不同氧浓度下进行体外培养其基因表达变化，实验结果表明猪胚胎发育效率在20% 浓度下培养的低于5%氧浓度下的原因在于，P66Shc基因通过调节ROS水平影响了猪胚胎体外发育。最后，研究了利用CRISPR/Cas9基因编辑方法构建敲除P66Shc基因猪胚胎的方法，通过构建该载体可以有效抑制P66Shc基因的表达，并且获得预期的结果。总之，目前已经研究发现了猪PP66Shc-ROS-胚胎发育调控作用轴的存在，能够影响猪胚胎体外发育的效率，这为后续建立高效的猪胚胎体外生产技术奠定了基础。