聚赖氨酸修饰丝素蛋白膜对神经干细胞生长和分化的影响

Effect of poly (L-lysine) modified silk fibroin film on the growth and differentiation of neural stem cells

摘要:

利用聚赖氨酸修饰丝素蛋白膜，观察其对神经干细胞 (NSCs) 生长及分化的影响，为中枢神经系统损伤修复材料的选择提供实验基础和理论依据。文中首先制备聚赖氨酸修饰的丝素蛋白膜，并通过核磁共振图谱和紫外-可见光谱进行验证。NSCs分别接种在单纯丝蛋白膜 (Silk)、聚赖氨酸修饰的丝蛋白膜 (Silk-PIL) 和多聚赖氨酸 (PLL) 上进行培养，分别在1、3、5、7 d时用CCK-8检测NSCs增殖活性。在第7天时，用免疫荧光染色检测NSCs分化情况，Western blotting和TUNEL检测细胞凋亡水平，Real-time PCR检测脑源性神经营养因子 (BDNF) mRNA水平。结果表明，核磁共振图谱和紫外-可见光谱证明聚赖氨酸成功地接枝到了丝素蛋白膜上，CCK-8检测显示：从第3天开始一直到第7天，NSCs在Silk-PIL上的增殖活性要显著高于Silk组 (P<0.05)，而与PLL组无显著性差异 (P>0.05)。免疫荧光观察显示，NSCs在Silk-PIL上分化成神经元的细胞显著多于Silk组 (P<0.05)，而与PLL组无显著性差异，3个组之间分化为星型胶质细胞的数量并无显著性差异。Western blotting 和TUNEL检测结果表明Silk-PIL组NSCs凋亡程度显著小于Silk组 (P<0.05)，但与PLL组无显著性差异 (P>0.05)。RT-PCR结果显示，NSCs在Silk-PIL和PLL组的BDNF mRNA表达水平显著高于Silk组 (P<0.05)。结果表明，聚赖氨酸修饰的丝素蛋白膜能够促进NSCs的增殖活性并减少NSCs细胞凋亡，同时促进NSCs向神经元方向分化，有望成为新型组织工程支架材料搭载NSCs移植修复中枢神经系统损伤。

Abstract:

In order to provide a basic theory for the materials of repairing central nervous system injury, we have studied the growth and differentiation of neural stem cells (NSCs) on poly (L-lysine) modified silk fibroin film. First, we used poly (L-lysine) to modify silk fibroin film and confirmed by UV–vis and 1H NMR spectra. Then NSCs were isolated and seeded on the silk fibroin film (Silk group), poly (L-lysine) (PLL group) and poly (L-lysine) modified Silk fibroin film (Silk-PIL group). The proliferation of NSCs was evaluated by Cell Counting Kit-8 (CCK-8) assay on days 1, 3, 5 and 7 after seeding. Immunofluorescence was used to analyze the differentiation of NSCs at the 7th day. The levels of apoptosis were detected by Western blotting and TUNEL. The mRNA level of brain derived neurotrophic factor (BDNF) was identified by real-time PCR. UV–vis and 1H NMR spectra confirmed that poly (L-lysine) was successfully grafted onto the silk fibroin film. From the 3rd day after seeding to the 7th day, the CCK-8 test showed that proliferation rate of NSCs in the Silk-PIL was significantly higher than Silk group (P<0.05) but had no significant difference compared with PLL group (P>0.05). Immunofluorescence staining displayed that more NSCs in Silk-PIL group were differentiated into neuron compared with Silk group (P<0.05), however, there was no significant difference compared with PLL group (P>0.05). The number of NSCs differentiated into astrocytes was not significantly different between the three groups. Western blotting and TUNEL test presented that the degree of apoptosis of NSCs in the Silk-PIL group was significantly lower than Silk group (P<0.05). RT-PCR exhibited that mRNA level of brain derived neurotrophic factor (BDNF) of NSCs was higher in Silk-PIL group compared with Silk group (P<0.05) but had no significant difference compared with PLL group (P>0.05). Thus, poly (L-lysine) modified silk fibroin film could promote the proliferation of NSCs and reduce NSCs apoptosis. Furthermore, it also can enhance the differentiation of NSCs into neurons. It is expected to become a new type of tissue engineering scaffold carrying NSCs to repair central nervous system injury.