MyoD诱导人胚胎干细胞向骨骼肌细胞分化治疗肌肉萎缩症的实验研究

Muscular dystrophies are a group of tissue degenerative diseases, so far there are no cures for this kind of genetic disorders. Repopulating the dysfunction tissue by transplantation of stem cells might be a potential therapeutic strategy, which require large amount and high purity of the transplantable cells. In this research, we use a lentiviral based Tet-on system to direct the differentiation of human embryonic stem cells (hESCs) into myogenic cells by control the expression of a skeletal muscle specific transcriptional factor: MyoD. By real time PCR and immunochemistry, we can check the expression pattern of different skeletal muscle related genes during differentiation. Then we use a myocyte calcium and contractivity recording system to identify the in vitro physiological function of the derived myotubes. Meanwhile, by transplantation of muscle injured NOD/SCID mice we can identify the in vivo function of the myogenic cells derived from hESCs. Finally, we can get the transplantable myogenic cells at a high purity (>90%). To our knowledge, it is the first time hESCs being directed into a kind of terminal differentiated cells by only one transcriptional factor. Moreover, this can be a foundation for future stem cell medicine research on muscular dystrophies.

肌肉萎缩症是一类目前无法治愈的组织器官退行性疾病，利用干细胞技术进行病变细胞的替代治疗是一种非常有希望的治疗方法。但是目前可用于肌肉萎缩症治疗的干细胞要么数量不多、要么成分复杂，难以达到干细胞治疗药物的要求。本课题拟利用慢病毒载体介导的Tet-on系统，可调控地表达骨骼肌发育过程中关键转录因子MyoD，诱导人胚胎干细胞（hESCs）进入骨骼肌细胞的分化程序。通过Real Time PCR和免疫化学染色等检测生肌过程中特异标记物的表达情况；并用NOD/SCID小鼠体内细胞移植实验、细胞内钙离子浓度与肌细胞收缩同步测定系统鉴定获得的生肌细胞和骨骼肌细胞的功能。最终得到高纯度（> 90%）、有功能的移植用细胞。本研究首次采用单一转录因子诱导hESCs分化为高纯度终末分化细胞。课题的完成将为研发治疗肌肉萎缩症的干细胞药物提供实验依据。

肌肉萎缩是由多种原因导致的组织器官退行性疾病，其后遗症之一瘫痪往往难于治愈。利用干细胞技术进行病变组织的修复治疗是一种非常有希望的治疗方法。但是目前可用于肌肉萎缩治疗的干细胞要么数量不多、要么成分非常复杂，难以达到干细胞治疗药物的要求。本研究利用慢病毒载体介导的Tet-on调控系统，可调控地表达骨骼肌发育过程中关键转录因子MyoD，诱导hESCs进入骨骼肌细胞的分化程序，建立了快速、高效的hESCs成肌细胞分化体系；并用该来源成肌细胞（纯度> 90%）移植治疗小鼠坐骨神经损伤造成的肌肉萎缩，证明该细胞移植治疗方法可以修复神经源性肌肉萎缩，包括形态和功能的修复。为研发治疗神经源性肌肉萎缩的干细胞药物提供了实验依据，具有较好的临床应用前景。