基于CNTF上调HCK激活STAT3促轴突再生探讨BMSCs治疗脊髓损伤的机理研究

脊髓损伤常导致严重神经功能障碍和患者终生残疾，而轴突再生是脊髓损伤后的难点。促进受损轴突再生可望成为脊髓治疗的有效手段。申请人前期发现，BMSCs可分泌CNTF并激活STAT3促轴突再生，从而改善脊髓损伤后神经功能缺损，但调控机制不清。预实验证实CNTF刺激促进脊髓神经元轴突生长，质谱分析发现CNTF处理后STAT3 Y705发生磷酸化，HCK高表达，给予CNTF同时下调HCK后神经元轴突再生受抑制。基于此提出假设：CNTF可能通过上调HCK，磷酸化激活STAT3构成BMSCs促轴突再生从而治疗脊髓损伤的作用机制。该项目拟从细胞、动物和分子水平①探讨HCK在促轴突再生中的功能；②明确HCK通过磷酸化激活STAT3介导BMSCs/CNTF促轴突再生；③CO-IP验证HCK可直接结合STAT3调控其活性的分子机制。最终为脊髓损伤研究提供新靶点，为BMSCs治疗脊髓损伤提供有力的实验依据。

Spinal cord injury (SCI) often leads to severe neurological dysfunction and lifelong disability, and axon regeneration is a difficult point after SCI. The regeneration promotion of the damaged axons is expected to be an effective mean of spinal cord therapy. The applicant previously found that BMSCs could secrete CNTF and activate the STAT3 signaling pathway to promote axon regeneration, thus improving the neurological deficit after SCI, however, the regulatory mechanism was unclear. Our preliminary experiment confirmed that CNTF stimulation promoted axonal growth in spinal cord neurons. Mass spectrometry analysis revealed that the 705th tyrosine site of STAT3 was phosphorylated after CNTF treatment, and the tyrosine kinase HCK was highly expressed; besides, the neuronal axon regeneration was inhibited when CNTF was given and HCK was downregulated. Based on the above information, we proposed the hypothesis that the mechanism of BMSCs promoting axon regeneration to treat SCI may be formed via CNTF upregulating HCK expression and phosphorylation activating STAT3. Thus, the project intends to explore the role of HCK activating STAT3 mediated BMSCs/CNTF in neuronal axon regeneration function; and to verify the molecular mechanism of HCK directly binding to STAT3 to regulate its activity by CO-IP in cells, rats and molecular levels. Our study may provide a new target for the study of spinal cord injury, and provides a powerful experimental basis for the clinical application of BMSCs in the treatment of spinal cord injury.