肌肉干细胞的干性保持调控机制研究

Muscle stem cells are the major source for postnatal muscle growth and muscle regeneration. These cells are activated from quiescent status upon injury or disease, then further go through proliferation and differentiation processes to form new functional myofibers. Part of the activated muscle stem cells can go back to the quiescent status and replenish the stem cell reservoir. How activated stem cells maintain their stemness and the abilities of rapid expansion remains to be elusive. Investigaton on the above questions has been hamperred by the lack of proper experimental system. The in vivo muscle stem cell activation and expansion are transient and difficult to trace; while muscle stem cells lose most of their stemness once cultured in vitro. We rescently established a long term expansion system to culture the activated muscle stem cells and maintain their stemness in vitro. In this system, we identified two groups of muscle stem cells with high or low ability to repair muscle injury in vivo, respectively, though they appear similarly in vitro. These findings provide us an exerllent system to investigate the stemness maintenance of muscle stem cells. We will perform RNA-seq, mass-spec, and other high throughput assays to identify factors (proteins and non-coding RNAs) critical for stemness maintenance, the functions of these factors will be further examined in cultured muscle stem cells and in vivo. The mechanism of how these factors regulating stemness maintenance and long term expansion will also be investigated.

肌肉干细胞负责出生后骨骼肌的生长和再生，在损伤或疾病诱导的肌肉再生过程中，肌肉干细胞由静息状态被激活，经过增殖、分化后形成新的肌纤维，部分增殖后的肌肉干细胞重新回到静息状态，回归干细胞库。在上述过程中，肌肉干细胞如何在激活后继续保持干性，同时大量扩增，目前所知甚少。由于激活的肌肉干细胞无法在体外长期培养条件下保持干性，而体内系统具有瞬时性和复杂性，难以进行系统的机制研究。申请人建立了能够长期维持激活的肌肉干细胞干性的体外培养系统，并在此过程中鉴定出两类具有不同整合能力的肌肉干细胞，为研究肌肉干细胞的干性保持提供了优良的实验系统。我们将利用此实验系统，通过RNA-seq、Mass-spec等方法鉴定与干性保持和活跃扩增有关的蛋白和非编码RNA，在肌肉干细胞中和体内验证这些相关因子的功能，并阐明其作用的分子机制。

本项目围绕关键科学问题“肌肉干细胞干性保持与扩增的关键调控因子鉴定”，鉴定TNC、Msi2、HuR、长非编码RNA编码的微肽LEAP等参与肌肉干细胞干性保持和分化的关键因子,阐明Vgll4调控干性保持与增殖以及分化之间的协同关系,并在此基础上建立了分离纯化人和猪肌肉干细胞的方法，以及体外培养的方法，发现表征人和猪肌肉干细胞干性的分子标记，为肌肉干细胞的再生医学应用奠定了基础。发表受本项目资助的论文12篇，其中IF>10论文4篇，参与撰写专著章节1篇。申请国家发明专利4项，其中授权1项，授权美国发明专利1项，日本发明专利1项；获批肌肉干细胞临床实验1项。培养博士研究生2名，硕士研究生1名，中国科学院青年科学促进会会员1名。项目按照原定计划执行，完成全部研究任务，超额完成所有考核指标，达到预期研究目标。