小鼠体细胞核移植胚胎发育中的自噬调控研究

Autophagy is a process by which cytoplasmic components including macromolecules and organelles are sequestered and degraded by lysosomal system. This process wildly exists among eukaryotes and is also a kind of intracellular refreshment system. Somatic cell nuclear transfer (SCNT) is now becoming one of the hottest fields in the biological research, which is significant and valuable in scientific application. However, there are still many problems, especilly epigenetic reprogramming, which lead to low efficiency of animal cloning. In recent years, some researches have reported that autophagy is required for reprogramming of somatic cells to form induced pluripotent stem cells (iPSCs). SCNT also requires resetting of the gene expression program of a somatic cell to a state consistent with embryonic development. So, autophagy may play a crucial role in genome reprogramming in SCNT. However, researches on the impacts of autophagy on preimplantation embryos development and genome reprogramming in SCNT were very limited until now. In current study, we will use mice as for model and try to find out the effects and systematic gene expression profile of autophagy during fertilized and SCNT preimplantation embryos development.Then, we will discuss the regulating effects of autophagy on promoting genome reprogramming in SCNT and try to explain how it works. These results will effectively promote the production of cloned animals.

自噬是普遍存在于真核细胞中的依赖溶酶体途径对自身结构的吞噬和降解的过程，也是一种细胞内的再循环系统。体细胞核移植是生物技术领域研究的热点之一,其科学意义和应用价值重大，但存在着以不完全重编程为代表的克隆效率低的问题。近年来有报道指出自噬在体细胞iPSC的诱导过程中有促进重编程的作用。体细胞核移植胚胎的构建同样需要使体细胞核去分化转变为未分化的过程，因此，自噬可能在体细胞核移植胚胎重编程过程中也有重要作用。但目前关于自噬在体细胞核移植胚胎发育和重编程中的作用未见研究报道。本项目以小鼠为模型，确定受精胚胎和核移植胚胎发育中的自噬生成和表达规律，探讨核移植胚胎发育中调控自噬对重编程效率的促进，解析自噬在核移植胚胎发育中的作用和重编程过程中的机制，为胚胎克隆技术的更好应用奠定基础。

自噬是真核细胞通过溶酶体降解自身受损的细胞器和大分子物质进而实现细胞内再循环的过程。哺乳动物中，自噬最早发生在受精卵，并对合子基因组启动、着床前胚胎发育有重要作用。近年来，体细胞核移植技术取得了很大进展，但供体核不完全重编程造成克隆效率低的问题制约了该技术的发展。为了确定自噬在核移植胚胎重编程过程中是否也有重要的调节作用，我们以小鼠为模型，比较了自噬标志性基因LC3在单精子注射、核移植和孤雌激活胚胎植入前各发育阶段中的表达，发现重构胚激活期间核移植和孤雌胚胎中没有自噬体的形成，而激活期间用自噬诱导剂-雷帕霉素处理能够产生自噬体。诱导自噬能够促进核移植胚胎中以5甲基胞嘧啶转换为5羟甲基胞嘧啶为代表的DNA去甲基化和母源性mRNA的降解。进一步，激活自噬后核移植胚胎的囊胚率与对照组相比得到显著提高（68.5% vs. 41.5%, P < 0.05）。进一步研究发现核移植和孤雌胚胎激活时添加的细胞松弛素B，抑制了微丝的聚集从而减少了自噬体的形成，在核移植和孤雌胚胎激活过程中去掉细胞松弛素B后发现能够产生自噬体。综上所述，自噬对核移植胚胎重编程和发育非常重要并且激活过程中自噬的抑制可能是导致克隆效率低下的重要原因。