CAJ | 학술논문

通过层粘连蛋白(LN)对壳聚糖-明胶-透明质酸-硫酸肝素(C-G-Ha-HS)复合支架进行修饰，提高支架粘附率，优化神经干/祖细胞(NS/PCs)三维培养体系，同时考察了原儿茶酸(PCA)对三维条件下NS/PCs分化为多巴胺(DA)能神经元的影响。扫描电镜观察及CCK-8分析表明，C-G-Ha-HS复合支架孔径为90~130μm，孔隙内的NS/PCs伸出类似神经样的突触使支架与细胞相互连接形成网状结构；培养4 h后，LN修饰的C-G-Ha-HS(5：5)支架细胞粘附率增加至未修饰组的(113.53±4.32)%；培养96 h后，细胞活率增加至未修饰组的(120.30±6.65)%；免疫细胞化学鉴定表明，1%胎牛血清(FBS)和0.06 mmol?L-1 PCA协同作用，明显提高了NS/PCs向DA能神经元分化率，TH阳性细胞分化率达到(16.53±0.65)%，增加至空支架组的(172.72±6.79)%；Nurr1阳性细胞分化率达到(15.93±0.91)%，增加至空支架组的(181.23±1.04)%；研究结果为PCA应用于NS/PCs移植治疗PD提供了实验依据。
통과층점련단백(LN)대각취당-명효-투명질산-류산간소(C-G-Ha-HS)복합지가진행수식，제고지가점부솔，우화신경간/조세포(NS/PCs)삼유배양체계，동시고찰료원인다산(PCA)대삼유조건하NS/PCs분화위다파알(DA)능신경원적영향。소묘전경관찰급CCK-8분석표명，C-G-Ha-HS복합지가공경위90~130μm，공극내적NS/PCs신출유사신경양적돌촉사지가여세포상호련접형성망상결구；배양4 h후，LN수식적C-G-Ha-HS(5：5)지가세포점부솔증가지미수식조적(113.53±4.32)%；배양96 h후，세포활솔증가지미수식조적(120.30±6.65)%；면역세포화학감정표명，1%태우혈청(FBS)화0.06 mmol?L-1 PCA협동작용，명현제고료NS/PCs향DA능신경원분화솔，TH양성세포분화솔체도(16.53±0.65)%，증가지공지가조적(172.72±6.79)%；Nurr1양성세포분화솔체도(15.93±0.91)%，증가지공지가조적(181.23±1.04)%；연구결과위PCA응용우NS/PCs이식치료PD제공료실험의거。
Chitosan-gelatin-hyaluronate-heparan sulfate (C-G-Ha-HS) scaffolds were fabricated and modified with laminin (LN) in order to improve scaffold adhesion and optimize 3-D culture system of neural stem/progenitor cells (NS/PCs). The effects of protocatechuic acid (PCA) on the inducing of NS/PCs differentiation into dopaminergic neuron were determined at 3-D level. SEM and CCK-8 assay results show that the pore size of the C-G-Ha-HS scaffolds is 90~130 μm and the NS/PCs are connected with the scaffolds to form reticular structure. Compared with LN-unmodified C-G-Ha-HS(5:5) scaffolds, LN-modified scaffolds enhance the NS/PCs adhesion rate by (113.53 ± 4.32)%after 4 hours and increase cell viability by (120.30 ± 6.65)%after 96 hours. Immunocytochemistry assay shows that the synergistic effect of 1%Fetal Bovine Serum (FBS) and 0.06 mmol?L-1 PCA can enhance the differentiation rate of NS/PCs into neurons. The differentiation rate of TH and Nurr1 positive cell can reach up to (16.53 ± 0.65)% and (15.93 ± 0.91)%, respectively. Compared with the control scaffold group, the differentiation rate of TH and Nurr1 positive cell are enhanced by (172.72 ± 6.79)%and (181.23 ± 1.04)%. These results provide experimental data for the application of PCA in the treatment of PD by NS/PCs transplantation.