组蛋白去乙酰化酶抑制剂联合处理对体细胞核移植的表观遗传水平的影响

Somatic cell nuclear transfer (SCNT) technologyis a widely used method for somatic cell reprogramming.However, the cloned embryos usually exhibit some epigenetic abnormalitiesbecause of the incomplete reprogramming. It has been proved that the cloning efficiency can be improved bytreating withthe histone deacetylation inhibitors (HDACis). However, in most cases, the epigenetic abnormalities observedin trophoblast ectoderm (TE) of cloned embryos which led to hypertrophy placenta in pups can not be rescued completely. But indeed we found that single HDACi could reduce the methylation level of TE at blastocysts stage. Here, we hypothesized thatthe treatment of cloned embryos with several HDACis at the same time mayfurther rescuethe epigenetic modifications in cloned embryos. In the present proposal we are using mouse as an animal model to address if the treatment of somatic cloned embryos withHDACi mixtures can indeed rescue the epigenetic abnormalities of cloned embryos which can eventuallyimprove the development of cloned embryos both in vitro and in vivo. To accomplish these goals, we will use molecularbiology, cell biology and reproductive biology techniques combined withsingle cell deep sequencingand stem cell techniquesto examine the outcomes oftreatment of cloned embryos withHDACicocktails. By accomplishing this study, we hope that the results mightprovide new ideas and feasible method for further improvement ofreprogramming efficiency in SCNT.

体细胞核移植(SCNT)胚胎因为重编程不完全导致在发育过程中出现各种表观遗传异常。我们已报道使用单一的组蛋白去乙酰化酶抑制(HDACi)，可以提高克隆动物制备效率，但是克隆胚胎滋养外胚层(TE)异常导致的胎盘肥大等仍然未得以改善。我们发现单一HDACi处理能一定程度降低TE的甲基化水平，对此，我们提出假设：将几种HDACi对克隆胚胎进行联合处理可能进一步使其表观遗传学水平趋向正常。为此，我们以小鼠作为模型，将不同种类和浓度的HDACi调制成Cocktail后处理克隆胚胎，并尝试建立克隆囊胚滋养层由来干细胞；通过免疫荧光染色，TUNEL染色，微量细胞深度测序，Q-PCR等手段，从胚胎，分子,干细胞等多方面探讨HDACi联合处理后对克隆胚胎发育的影响。本项目有望利用各HDACi功能上的互补修复克隆胚胎表观遗传水平的异常，提高克隆动物制备效率，为SCNT技术的发展提供全新的思路和可行的方法。

体细胞核移植技术对于研究动物胚胎发育过程、体细胞进行重编程的机制以及将基础科研转化为再生医学领域的研究都起着重要的意义。通过十多年的研究，大量证据都表明该技术效率低下的根本原因在于供体细胞特异性的组蛋白修饰无法完全重编程，导致了在克隆胚胎在发育的早期阶段，组蛋白的乙酰化水平都与正常的受精卵出现了很大的差异。因此，探索和发现克隆胚胎早期发育阶段组蛋白修饰的异常，寻找表观遗传水平异常出现的根源，针对性地修复表观遗传水平异常，成为该技术能否得以继续发展的关键所在。本项目将几类被证明有效的组蛋白去乙酰化酶抑制剂对克隆胚胎进行联合处理有望进一步使其表观遗传学水平趋向正常。本项目研究结果中得出TSA，Scriptaid，SAHA，Oxamflatin等四种组蛋白去乙酰化酶抑制剂不同的浓度的混合液在长时间处理后，克隆胚胎的囊胚发育能力超过70%，比未添加的对照组显著提升。将克隆胚胎移植回代孕母鼠后，其出生率高于使用单一组蛋白去乙酰化酶抑制剂2倍以上，揭示了小鼠克隆在组蛋白去乙酰化酶抑制剂联合处理后率能被维持在一个较高的效率。当将克隆囊胚种植到胚胎干细胞建系培养基后，克隆胚胎干细胞的建系效率高达78%，远远高于未处理克隆胚胎约30%的建系效率。综上所述，本项目将各HDACi功能上的互补大幅提高克隆动物制备效率，为体细胞克隆技术的发展提供了全新的思路和可行的方法。