基于Wnt信号通路探讨脱细胞脊髓支架与脂肪干细胞共建修复脊髓损伤的作用及机制

Spinal cord injury (SCI), an irreversible nerve damage caused by factors like trauma, can lead to severe dysfunction of limbs under the injured segments. However, there is no exact method to cure this disease at present. Previous studies showed that co-construction of silk fibroin/ chitosan scaffolds (SFCS) and adipose-derived stem cells (ADSCs) plays a decisive role in the repair of rats SCI, but with a limited effect. One reason is that as the signaling pathways through which ADSCs differentiate into neurons are not yet clear, it is impossible to promote differentiation of stem cells towards neurons by regulating signaling pathways. Another reason is that the failure of SFCS in simulating the three-dimensional structure of spinal cord tissue results in disordered axonal growth. Therefore, this project plans to repair rat SCI through con-construction of ADSCs with acellular spinal cord scaffolds which serve as the carrier. The main contents are as follows: 1. exploring the effect of the co-construction system of ADSCs and acellular spinal cord scaffolds to the differentiation of ADSCs towards neurons, and the effect of the Wnt signaling pathways in the differentiation process; 2. exploring the in-vivo role of co-construction system of ADSCs and acellular spinal cord scaffolds in the rats SCI model, and the effect of the Wnt signaling pathways in repair of SCI. The completion of this project will be favorable for further understanding of the molecular mechanism of repair of SCI, promote the research progress of spinal cord injury, and provide the possibility of improving the living quality and reducing the medical cost of patients.

脊髓损伤是由创伤等原因引起的不可逆性神经损害，导致损伤节段以下肢体严重的功能障碍，目前尚无特效的治愈手段。前期研究表明丝素/壳聚糖支架共载脂肪干细胞有助于大鼠脊髓损伤的修复，但作用有限，一方面脂肪干细胞向神经元分化的信号通路尚不清楚，无法通过调控信号通路来促进干细胞向神经元分化；另一方面丝素/聚糖支架无法模拟脊髓组织的三维结构，导致轴突生长无序。因此，本课题拟采用脱细胞脊髓支架作为载体与脂肪干细胞共建修复大鼠脊髓损伤，主要内容有：1.研究脂肪干细胞与脱细胞脊髓支架共建体系对脂肪干细胞向神经元定向分化的影响及Wnt信号通路在分化过程中的作用；2.研究脂肪干细胞与脱细胞脊髓支架共建体系对大鼠脊髓损伤模型的体内作用及Wnt信号通路在脊髓损伤修复中的作用。本项目的完成将有助于进一步理解脊髓损伤修复的分子机制，推进脊髓损伤的研究进展，为临床上提高患者生活质量、降低患者医疗花费提供可能。

背景：脊髓损伤是由创伤等原因引起的不可逆性神经损害，导致损伤节段以下肢体严重的功能障碍，目前尚无特效的治愈手段。前期研究表明丝素/壳聚糖支架共载脂肪干细胞有助于大鼠脊髓损伤的修复，但作用有限，一方面脂肪干细胞向神经元分化的信号通路尚不清楚，无法通过调控信号通路来促进干细胞向神经元分化；另一方面丝素/壳聚糖支架无法模拟脊髓组织的三维结构，导致轴突生长无序。因此，本课题拟采用脱细胞脊髓支架作为载体与脂肪干细胞共建修复大鼠脊髓损伤，并探讨Wnt通路在损伤修复过程中的作用，深入研究脊髓损伤修复的机制，对于提高脊髓损伤的修复效果有着重要意义。方法：采用胶原酶消化法分离获得的脂肪干细胞，向神经方向诱导分化，并验证Wnt通路在分化过程中的作用。构建脱细胞脊髓支架并采用扫描电镜检测其基本性能。脂肪干细胞接种于脱细胞脊髓支架上，通过测试粘附率和增殖率，评估二者生物相容性。研究脊髓损伤提取液对脂肪干细胞分化及神经营养因子表达影响的规律。研究脱细胞脊髓支架联合脂肪干细胞修复脊髓损伤的规律，并探讨Wnt通路在修复过程中的作用。结果：获得的脂肪干细胞具有多向分化潜能，Wnt信号通路在脂肪干细胞向神经元分化过程中起着重要作用。脱细胞脊髓支架和脂肪干细胞在体外共培养5d最佳，细胞增殖率和粘附率最高。脊髓损伤提取液主要通过影响微环境中NT-3的改变，来影响干细胞向神经元分化。脱细胞脊髓支联合脂肪干细胞通过抑制炎症反应、抑制细胞凋亡，降低神经元死亡、促进神经纤维生长来发挥抗脊髓损伤的作用，抑制Wnt通路后，上述修复作用明显减弱。结论：脱细胞脊髓支联合脂肪干细胞对于脊髓损伤的治疗有着积极的意义，Wnt通路在整个修复过程中起着重要作用。本项目的完成将有助于进一步理解脊髓损伤修复的分子机制，推进脊髓损伤的研究进展，为临床上提高患者生活质量、降低患者医疗花费提供可能。