牙鲆肌细胞增生和增肥的分子调控机制

Hyperplasia (cell number increase) and hypertrophy (cell size increase), which involving the development of muscle satellite cells (SC), are two primary forms of muscle growth. Due to their unclear mechanisms, the accurate target cannot be found during the process to get fast-growing fish. We will use diploid and triploid flounder SC as cell hyperplasia and hypertrophy model, respectively, and study their mechanisms by analyzing the difference between these two SC during the process of cell number doubling. Our preliminary results showed that cell number doubling time for the triploid SC was longer than that of diploid SC and the reason might be that the triploid SC is easier to activate and differentiate than the diploid SC. We will validate this hypothesis and test if the process for cell number doubling is the proliferation after SC activation through gene expression and cell proliferation analysis. Through RNA-seq, we will get the differentially gene expression profile in diploid and triploid SC using samples that before and after their activation. By comparison of these two different expression profiles, the unique and common differentially expressed genes will be got. The differentially expressed signaling pathways will be constructed. Then, the function of genes and signaling pathways will be further performed through overexpression/activation and knockout/knockdown/inhibition in these two SC. The molecular mechanisms of SC activation and differentiation, and the effects of ploidy will be clarified. Then, we will verify their function in cell hyperplasia and hypertrophy through activating and/or inhibiting related genes and signaling pathway in the muscle of juvenile flounder. Thus the molecular mechanism of cell hypertrophy and hyperplasia can be explored. This will provide a new theory for muscle growth, and it will provide new idea for getting fast-growing fish.

肌卫星细胞(SC)参与的肌细胞增生(数目增加)和增肥(体积增大)是鱼类肌肉生长的主要方式，它们调控机制的不明确致使人们在获取快速生长鱼时无法找到精准的靶点。我们将以牙鲆二倍体和三倍体SC为增生和增肥的模型,通过分析它们细胞倍增过程中的差异来解析增生和增肥的机制。初步实验显示三倍体SC较二倍体易激活、易分化可能是其倍增时间较长的原因，我们将通过基因表达及细胞增殖等实验确认该推测及细胞倍增的方式为SC激活后的细胞增殖；通过转录组数据分析比较二、三倍体SC激活前后的基因表达谱，获得差异基因，构建信号通路；通过在这两种SC中过表达/激活、敲除/敲降/抑制相关基因研究基因和信号通路的功能，阐述SC激活、增殖/分化的分子机制及染色体加倍的影响；在幼鱼中激活、抑制相关基因和信号通路，验证它们在细胞增生和增肥中的作用，从而形成相关理论。这将为肌肉生长机制提供新的理论，从而为获得快速生长鱼类的提供新思路。

鱼肉（骨骼肌）的生长是鱼类生长的主要表现形式，也是鱼类养殖业中重要的经济指标之一。肌肉生长主要是通过肌细胞增殖的增生过程及肌细胞融合等的增肥过程完成的，这两个过程都由肌卫星通过激活、增殖及分化过程参与，但是这两个过程之间的关系及调控并不是很清楚，本项目期望通过牙鲆中的研究解析相关的分子机制。本项目通过免疫荧光筛选了肌卫星细胞标志因子pax3/7的特异性抗体；通过组织学分析了牙鲆二倍体和三倍体混合养殖过程中各自肌纤维数量、直径的变化及两者的差异；通过组织学和qRT-PCR研究了1和2年龄牙鲆二倍体和三倍体鱼中肌纤维数量、直径与激活/分化及增殖相关因子表达的差异；通过MTT及RT-PCR分析了BMP信号通路对牙鲆胚胎肌卫星细胞增殖及分化的影响；对电转、显微注射等胚胎外源基因导入方法及基因编辑技术进行了探索及优化；通过原位杂交分析了肌细胞发育相关基因的表达特征；通过基因编辑技术探究了肌细胞融合基因myomaker的功能。通过上述研究获得了牙鲆肌卫星细胞的标志因子pax3/7的特异性抗体，为牙鲆肌卫星细胞的鉴定奠定了基础。在二倍体和三倍体混合养殖过程中，它们的体重增长率、肌纤维数量及直径的变化率没有显著差异；1和2年龄三倍体中肌细胞激活/分化因子myod的表达高于同龄二倍体的，单位面积肌纤维数量及新形成肌纤维明显少同龄二倍体的，因此二倍体肌细胞易增殖、三倍体的肌细胞易分化。BMP信号通路的抑制剂能够抑制胚胎肌卫星细胞中alk2/3的表达；建立了适合牙鲆卵显微注射用注射针的制备方法、显微注射方法及基因编辑技术平台，突变率在40%以上；获得了带有myomaker突变的嵌合体仔鱼，该仔鱼的每个快肌纤维的细胞核数量大多为1个，而对照中的数量为3个，这为在牙鲆细胞和活体中研究基因的功能奠定了基础。我们的研究结果将为阐述肌卫星细胞参与肌纤维增生和增肥的理论奠定基础。