牙鲆Pax3a和Pax3b选择性剪接体在肌细胞增殖和分化中的作用机制研究

Fish skeletal muscle，i.e., fish meat, is not only an important source of high-quality protein for humans, but also the purpose of fish farming. Muscle precursor cells (MPCs) are the source of muscle fibers during embryonic development. Pax3 gene, a specific marker of MPCs, is responsible for maintaining the proliferative ability and undifferentiated state of MPCs. In mammals, Pax3 is required for the formation of hypaxial muscles of the trunk and for the delamination and migration of MPCs to other sites of myogenesis such as those in the limb. Nevertheless, the role of Pax3 gene in the fish (especially marine fish) myogenesis is still little known. Olive flounder (Paralichthys olivaceus) is an important commercially cultured marine flatfish in China. In our preliminary experiments, we identified multiple alternative splicing isoforms of Pax3a and Pax3b genes in olive flounder, namely Pax3a-1, Pax3a-2, Pax3b-1, Pax3b-2 and Pax3b-3, however, the effect and molecular mechanism of these isoforms in muscle cells proliferation and differentiation is still poorly understood. This project aims to study their regulatory roles in muscle cells proliferation and differentiation. In this project, we will make use of nuclear localization analysis, transcriptional activity analysis, and interaction anylysis at different levels by using mammalian cell lines, flounder muscle cell line and zebrafish embryos. This project will deepen our understanding of the molecular mechanism reguating muscle cell proliferation and differentiation. This anticipated results may have importance in fish meat production.

鱼类骨骼肌即鱼肉是优质的蛋白源，也是鱼类养殖的追求目标。胚胎期肌肉前体细胞（muscle precursor cells, MPCs）是肌纤维的来源，Pax3表达是MPCs的特异性标志，负责维持MPCs的增殖能力和未分化状态，在哺乳类胚胎早期生皮肌节的侧部肌肉细胞至躯干和四肢的迁移中发挥重要作用。尽管如此，鱼类尤其海水鱼类Pax3基因在肌肉发生中作用机制的研究几乎未见报道。牙鲆是我国重要的海水养殖鱼类，我们从牙鲆中获得Pax3a和Pax3b基因及多种选择性剪接体，但它们在肌细胞增殖和分化中的作用机制尚不清楚。本项目拟利用哺乳类细胞系、牙鲆肌细胞系和斑马鱼胚胎活体实验，通过核定位、转录活性、相互之间的作用来研究牙鲆Pax3a/3b选择性剪接体对肌细胞增殖和分化的调控，探索海水鱼类MPCs标记基因Pax3的作用机制，将有助于我们获得相关的技术方法来促进肌肉生长，在水产养殖中增加鱼类肌肉产量。

该项目以我国重要海水养殖鱼类牙鲆为对象，对鱼类胚胎期肌肉前体细胞（muscle precursor cells, MPCs）标记基因pax3 的多种选择性剪接体（pax3a-1、pax3a-2、pax3b-1、pax3b-2 和pax3b-3）进行研究，不仅对比分析了各剪接体的亚细胞定位、对目的基因myod 的转录活性及其相互作用，而且初步研究了各剪接体在肌细胞增殖和分化中的作用。利用亚细胞定位分析发现pax3a-1、pax3a-2 和pax3b-1 均具有核定位功能，而pax3b-2 和pax3b-3 均不具有核定位功能，该结果与软件预测核定位信号结果一致。利用双荧光素酶报告基因检测系统分析了各剪接体对myod 的转录活性，发现pax3a-1、pax3a-2 和pax3b-1 均具有转录激活myod 的活性， pax3b-2 能够抑制myod 的转录活性，而pax3b-3 对myod 没有转录活性，pax3b-3 能够增强pax3b-1 对myod 的转录活性。利用细胞转染技术检测了各剪接体对C2C12 成肌细胞增殖和分化的影响，发现它们没有明显的调控作用，这可能受转染效率的影响。斑马鱼胚胎活体实验显示各剪接体在胚胎早期肌肉发育-肌细胞发生过程中具有抑制作用，但在胚胎晚期肌肉发育过程中，pax3a-2 和pax3b-1 却有促进分化的作用，牙鲆幼鱼活体实验也显示其对晚期肌肉发育影响不大。同时，我们还拓展了相关的研究，分析了孵化期和成体期牙鲆侧线处骨骼肌纤维的组成特征，发现成体期牙鲆表层肌肉是快肌和慢肌的嵌合体；以pax3a 和pax3b 为早期胚胎神经和肌肉发育的指示因子，初步探讨了人工染色体操作对牙鲆胚胎早期发育影响的机制；分离培养并建立了牙鲆胚胎期肌卫星细胞系；从斑马鱼中鉴定出一个具有正向调节快肌分化的基因DYRK2；分析了牙鲆神经肽Y对牙鲆生长的影响。这些研究丰富了我们对鱼类肌肉发育和调控的认识，将有助于鱼类肌肉发育机制的解析。