低强度脉冲超声激励iPS诱导的神经嵴干细胞移植治疗脊髓损伤的实验研究

The rate of morbidity and disability of spinal cord injury (SCI) is high and thus currently the treatment of SCI concentrates on cell transplantation therapy. However, it is difficult for the transplanted "seed cells" to survive and differentiate in the inhibitory micro-environment after SCI, therefore how to obtain "seed cells"with high viability is a big issue. The previous work shows that the low-intensity pulsed ultrasound（LIPUS）can significantly enhance the viability, proliferation and differentiation of cells, however,the best intervention methods and molecular mechanism, as well as signal pathways, are still not clear and to be investigated. The project is to explore the best LIPUS intervention on the basis of experimental research and modeled by artificial intelligence to improve the viability of "seed cells" and discover the molecular mechanisms. Meantime the experimental system is to be optimized in terms of both hardware and software in order to be more suitable for project experiments with cells. Neural crest stem cells (NCSCs) are better than other pluripotent stem cells in terms of differentiating to neurocytes. Therefore it is purposed to employ neural crest stem cells differentiated from induced pluripotent stem cells (iPSCs), with higher viability due to intervention by LIPUS,to be transplanted for treating SCI. The observation of the effect of iPSCs-NCSCs, intervened by LIPUS in the treatment of spinal cord injury, provides new ideas to achieve effective and early rehabilitation after spinal cord injury.

脊髓损伤（SCI）发病率高、致残率高，细胞移植治疗是目前的研究热点。而移植的"种子细胞"在SCI局部抑制性微环境难以存活并分化，因此获得高活性的"种子细胞"是急需解决的问题。课题组前期实验发现，低强度脉冲超声（LIPUS）能显著增强细胞的活性及增殖分化能力，而其最佳的干预方法及潜在的分子机制和信号通路并不清楚。本课题将以实验研究为基础，通过人工智能方法建模深入探索LIPUS干预的最佳方法以提高被移植细胞活力，进一步阐述其潜在的分子机制；同时从硬件和软件两方面进一步优化超声实验装置，使其能更适用于细胞实验。神经嵴干细胞（NCSCs）较其他多能干细胞具有更强的向神经细胞分化能力，因此本课题拟应用诱导多能干细胞（iPSCs）分化成的NCSCs，在LIPUS干预后将高活性iPSCs-NCSCs移植治疗SCI，观察其治疗效果和细胞增殖分化情况，为实现脊髓损伤的有效修复和早期康复提供新的思路。

脊髓损伤（spinal cord injuries, SCI）是骨科一种严重的创伤性疾病，其发病率高，致残率高，目前尚无有效治愈的方法。干细胞移植治疗，已被认为是针对脊髓损伤患者运动和神经的恢复最有希望的一种治疗方法。而移植的“种子细胞”在SCI 局部抑制性微环境难以存活并分化，因此获得高活性的“种子细胞”是急需解决的问题。低强度脉冲超声（Low intensity pulsed ultrasound, LIPUS）已被广泛应用于基础研究和临床应用，本研究通过利用自主搭建的低强度脉冲超声干预系统，输出不同强度超声以激励iPSCs-NCSCs，检测iPSCs-NCSCs 增殖活性改变。以实验研究为基础，通过人工智能方法建模深入探索LIPUS 干预的最佳方法以提高被移植细胞活力,并得到LIPUS 最佳干预条件，获得优化LIPUS-iPSCs-NCSCs 种子细胞。体外实验评价了LIPUS干预的安全性，并对营养因子的分泌以及LIPUS干预后Notch信号通路的改变进行检测。此外，课题组团队将优化的iPSCs-NCSCs进行细胞移植治疗。然后对大鼠进行后肢运动功能进行评价，并进行免疫组织化学和组织学观察，以说明其对脊髓损伤的修复机制。.通过实验，本课题组发现用LIPUS激励后的iPSCs-NCSCs在体外获得了更高的细胞活力，LIPUS促进了神经营养因子的表达，但并不影响细胞的凋亡。在脊髓损伤后进行细胞移植治疗发现：LIPUS-iPSCs-NCSCs移植组大鼠运动功能得到明显改善，脑源性神经营养因子（BDNF）和神经生长因子（NGF）等神经营养因子的表达明显上调。组织学观察显示，与其他实验组相比，LIPUS-iPSCs-NCSCs移植组的空洞面积明显减少。.通过以上实验课题组团队认为LIPUS可以提高体外培养的神经嵴干细胞活力及神经营养因子的表达，优化的iPSCs-NCSCs移植治疗可以获得更好的功能恢复。低强度脉冲超声作为一种无创的物理干预方式，为细胞移植修复脊髓损伤的种子细胞优化提供了一种新的手段。