用miR-23~miR-27~miR-24基因簇双敲除小鼠研究其在心脏发育中的功能

The heart is the first formed organ during the during mammalian embryogenesis, on the fundamental ,all life activities and organism was conducted of. The structure formation and playing function of the heart mainly rely on a precise spatiotemporal control of a set of signaling pathways network. Many factors that influence and disturb the network can cause serious heart disease. microRNAs, especially microRNA cluster family, now constitute an important component of the regulatory circuits in heart developmen process relyed on its unique post transcriptional regulation mechanism. However, with the human genome encoding over 1000 miRNAs, only a little of them have been studied in the heart, and most of the largest and most abundant cardiac miRNA families have not yet been extensively characterized, because of the technical difficulties for complete genetic deletions of multiple miRNA family members located on multiple different chromosomes..Mouse miR-23~27~24 clusters, which locate on two chromosomes as miR-23a~27a~24-2 and miR-23b~27b~24-1 cluster separately. Their analogous functions in ESCs and similar sequence at the seed region suggest that the members may be functional compensative or redundant. In our previous research, we generated miR-23a~27a~24-2 cluster (miR-23a cluster) and/or miR-23b~27b~24-1 cluster (miR- 23b cluster) bi-allelic knockout ESC clones mediated by CRISPR/Cas9 technology, and elucidated that the miRNA clusters were indispensable for ESC mesoderm differentiation in vitro and in vivo. .In this study,we will construct miR-23~miR-27~miR-24 cluster family double knockout mouse and determine the single and double knockout mouse ‘s carciac development status in the whole animal,cell and molecular levels. Through researching in the mechanism of this microRNA cluster family’s target genes and signal pathways in heart development, we can elucidate the interaction of this family’s different cluster function. This work will provide more comprehensive information and evidence for us to understand the microRNA related functions in the development of heart and regulation mechanism in the network . We believe that these microRNA cluster family play an important role in heart development, thus addi¬tional studies may clarify the mechanism(s) of normal heart development, and provide a physiological basis for future investigations on heart disease and give new cues for the development of new therapeutic method of cardiac disease .

心脏是胚胎发育过程中形成的第一个组织器官，而后有机体的所有生命活动均在该基础上进行。心脏的形成和功能发挥主要依靠一套精细的信号调控网络，影响和干扰这个网络都会引起严重的心脏疾病，microRNA尤其是成簇microRNA，在这个网络中起着极为重要的调节作用。由于许多同源的成簇microRNA位于不同的染色体上，在技术上限制了其研究。在我们的前期工作中，通过CRISPR/Cas9介导的方法获得同源的miR-23a/23b基因簇双敲除的小鼠胚胎干细胞，发现其有严重的中胚层分化障碍。在本研究中，我们拟构建miR-23a/23b双基因簇敲除小鼠，观察其心脏发育状况，从动物整体、细胞到分子水平阐明该基因簇调节心脏发育的作用机制，为我们理解相关microRNA在心脏发育调控网络中的作用机制提供更加全面的信息和证据，加深对microRNA网络在心脏疾病发生发展中的认识，为治疗心脏疾病开发新的治疗手段。

心脏是胚胎发育过程中形成的第一个组织器官，其形成和功能发挥主要依靠一套精细的信号调控网络，microRNA尤其是成簇microRNA家族，因其独特的转录后调控机制在这个网络中起着极为重要的调控作用。由于许多同源的成簇microRNA位于不同的染色体上，在技术上限制了其研究。在我们的前期研究中，通过CRISPR/Cas9介导的方法获得同源的miR23a/23b基因簇双纯合敲除（miR23a-/-/ miR23b-/-）的小鼠胚胎干细胞（ESC），发现其有严重的中胚层分化障碍。我们利用miR23a+/-/23b+/- ESC构建了嵌合体小鼠，在嵌合体不能进行种系传递的情况下，通过CRISPR/Cas9技术，分别得到miR23a+/-及miR23b+/-小鼠。通过不同基因型小鼠间的合笼，已经得到miR23a-/-、miR23b-/-和miR23a-/-miR23b-/-小鼠，其后代仔鼠的基因型分布均符合孟德尔遗传定律，未观察到双基因敲除小鼠导致胚胎致死的情况，性成熟前其体重及健康状况未观察到明显异常。但是，miR23a-/-/miR23b-/-双纯合敲除小鼠从3月龄开始出现异常，如行动迟缓、毛发稀疏等，严重的会导致死亡；另外母鼠可以正常怀孕但无法正常分娩。同时，我们注意到Journal of Biological Chemistry杂志在2018年发表的一篇文章指出miR23a纯合敲除会导致小鼠胚胎致死，这与我们所观察到的现象不一致，我们调整了研究方案，将研究内容聚焦到了骨骼及心脏检测，将进一步通过绘制生长曲线、血常规及血生化分析、心脏超声、骨密度检测、检测不同脏器组织学异常以及收集DNA、RNA和蛋白样品研究相关信号通路的变化，力争尽快完成相关研究工作。