骨骼肌干细胞在神经慢性卡压后的变化及调节机制的研究

Chronic nerve compression could lead to muscle atrophy and fibrosis. Some studies have demonstrated that fibrosis in denerv ated skeletal muscle is related with skeletal muscle stem cells (SMSCs). Although SMSCs could be harvested after three weeks in culture condition, the molecular features of SMSCs after culturing might be different than SMSCs in nerve compression muscle. In this research project, we are going to collect SMSCs from nerve compression muscle directly by fluorescence activated cell sorting (FACS). Molecular and functional properties of SMSCs from nerve compression muscle will be investigated and compared with SMSCs from intact muscle. Microarray and PCR-array approaches will be used to characterize molecular features of SMSCs from nerve compression muscle. Furthermore, the data from in vitro condition will be verified by in vivo experiments. Genes highly expressed in SMSCs will be analyzed in nerve compression muscle by fluorescence in situ hybridization (FISH) and immunohistochemistry. The preliminary experimental results from our lab showed that TGF-beta and Notch signaling pathway components were much more expressed in SMSCs from nerve compression muscle than ones in intact muscle, implying TGF-beta and Notch signaling pathway activated in SMSCs from nerve compression muscle. If these results are proved in this project, these signaling pathways will be activated or inhibited in vivo to identify the role these classic pathways for regulating SMSCs in nerve compression muscle. Additionally, other signaling cascades and factors will be explored as well if their expression levels are impressively changed in SMSCs during nerve compression. Finally, conditional cell ablation mouse model will be used to identify the role of SMSCs during chronic nerve compression. In conclusion, we are going to explore the characteristics of SMSCs in nerve compression muscle, and then investigate the regulatory network for SMSCs in this condition.

慢性神经卡压是常见的周围神经疾病，其可致骨骼肌萎缩、纤维化以及肢体功能障碍。一直以来，慢性神经卡压动物模型存在个体差异较大、卡压程度难以量化等问题。最近，本实验室成功建立了药物诱导的大鼠坐骨神经卡压模型，其方法稳定、有效，构建过程相对简单。在此基础上，本实验室将通过侧群细胞分离方法收集骨骼肌干细胞，使用Microarray、PCRarray、原位杂交技术等了解骨骼肌干细胞在卡压过程中及卡压松解后的基因表达变化情况。进而，以此为依据，结合本实验室的前期研究结果，探索影响骨骼肌干细胞行为变化的调节机制。最后，我们将使用条件性细胞敲除模型明确骨骼肌干细胞在慢性神经卡压过程中的角色与作用。本研究的意义有以下三点：⑴ 提供了一种高效、稳定的大鼠坐骨神经卡压模型;⑵ 系统探索了骨骼肌干细胞在神经卡压后的调节机制;⑶ 明确了骨骼肌干细胞在神经卡压导致的骨骼肌萎缩过程中的作用。

慢性周围神经卡压所造成的神经功能障碍、支配区域肌肉萎缩等问题一直是临床工作中的一个难点，国内外对于其病变机制的实验研究也是鲜有报道。本项目首先创建了一种新型的慢性周围神经卡压动物模型，并采用多种方法证实其稳定性和有效性，然后在此基础上探索了支配肌肉在慢性神经卡压以及解除卡压后的动态变化过程，发现远端的小肌肉相较于近端肌肉，更容易受慢性周围神经卡压的影响，解卡压后近端肌肉较远端肌肉的恢复效果更好，且早期解卡压肌肉的恢复效果明显好于晚期解卡压。另外，本项目深入探索了骨骼肌失神经萎缩的分子机制，证实了TGF-β1对骨骼肌的促萎缩作用，同时发现自噬（autophagy）在骨骼肌失神经萎缩早期的明显活化，并结合转录组测序技术发现高迁移率族蛋白B1（HMGB1）在萎缩肌肉中的高表达，然后通过通路抑制及激活手段，进一步证明了HMGB1/autophagy信号通路在TGF-β1促骨骼肌萎缩过程中的关键作用。综上所述，本项目在研究过程中建立了一种稳定、有效的慢性周围神经卡压模型，为慢性神经卡压的实验研究奠定了坚实基础，并探索了卡压及解卡压后支配骨骼肌的相应变化，同时深入研究了骨骼肌失神经萎缩的分子机制，这为临床治疗及改善骨骼肌萎缩问题提供了重要的参考思路。