细胞外基质刚度调控压电型机械敏感离子通道组件1对神经干细胞分化的影响

Extracellular Matrix Stiffness Affects Differentiation of Neural Stem Cells by Regulating Piezo1

目的 本研究旨在探讨细胞外基质刚度变化对神经干细胞(neural stem cells,NSCs)分化的影响及其作用机制。方法 本研究基于成功构建脊髓损伤大鼠模型,并制备不同刚度(0.7 k Pa、40 k Pa)的聚丙烯酰胺凝胶基底,将大鼠原代NSCs于不同刚度基底上培养。压电型机械敏感离子通道组件1 (piezo type mechanosensitive ion channel component 1,Piezo1) sh RNA质粒转染NSCs细胞。免疫荧光染色检测神经元标志物双皮质醇(doublecortion,DCX)和星形胶质细胞标志物胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)阳性细胞百分比。免疫组织化学及蛋白质免疫印迹(Western blot)法检测损伤组织及NSCs细胞中Piezo1蛋白的表达水平。结果 与0.7 k Pa基质刚度组相比,40 k Pa基质刚度组中DCX阳性细胞数增加,而GFAP阳性细胞数减少,Piezo1蛋白表达量上升。脊髓损伤大鼠损伤组织Piezo1蛋白表达显著高于空白对照(sham)组。40 k Pa基质刚度条件下沉默Piezo1后,DCX阳性细胞数减少,而GFAP阳性细胞数增加,差异具有统计学意义(P<0.05)。机制研究发现,沉默Piezo1导致IV型胶原及纤连蛋白表达下降。重组纤连蛋白逆转了Piezo1 sh RNA对NSCs分化的影响,即DCX阳性细胞数增加,而GFAP阳性细胞数减少。结论 综上可见,硬基底刚度通过促进Piezo1蛋白表达,上调IV型胶原及纤连蛋白表达,从而调控NSCs细胞分化。本研究为基于生物材料治疗脊髓损伤提供了新的视角。

Objective The aim of this study is to investigate the effect of matrix stiffness changes on the differentiation of neural stem cells(NSCs) and the underlying mechanism. Methods A rat model of spinal cord injury was constructed. Polyacrylamide gel substrates with different stiffness(0.7 k Pa, 40 k Pa) were prepared, and were cultured with primary rat NSCs. NSCs cells were transfected with piezo type mechanosensitive ion channel component 1(Piezo1) sh RNA plasmid. Immunofluorescence staining was used to detect the percentage of positive cells for the neuron marker doublecortion(DCX) and the astrocyte marker GFAP. Immunohistochemistry experiment and Western blot were used to detect the protein expression of Piezo1. Results The results showed that compared with the 0.7 k Pa group, the number of DCX-positive cells in the 40 k Pa group increased, while the number of GFAP-positive cells decreased, as well as the protein expression of Piezo1 increased. The expression of Piezo1 was significantly up-regulated in the injured tissue of rats with spinal cord injury compared with the sham group. Silencing of Piezo1 reverse the effect of 40 k Pa matrix stiffness on NSCs proliferation, as indicated by decreased number of DCX-positive cells, and increased number of GFAPpositive cells increased. Further studies found that knockdown of Piezo1 leads to decreased expression of collagen IV and fibronectin. Recombinant fibronectin reversed the effect of sh-Piezo1 on the differentiation of NSCs. Conclusion Rigid base stiffness regulates the differentiation of NSCs by promoting the expression of Piezo1 protein and up-regulating the expression of type IV collagen and fibronectin. This study provides a new perspective for the treatment of spinal cord injury based on biomaterials.