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目的:建立体外神经干细胞(neural stem cells,NSCs)迁移研究的平台,并通过经典名方补阳还五汤对此平台进行适用性验证。方法:分离、培养大鼠脑胚胎NSCs,利用NSCs放射性迁移、划痕修复动态检测以及Transwell趋化性迁移检测的方法,建立体外NSCs迁移研究平台。利用此平台评价经典名方补阳还五汤(300,600 mg·L~(-1)),有效单体川芎嗪(10,50 mg·L~(-1))以及阳性对照基质细胞衍生因子-1(stromal-derived factor 1,SDF-1)等对NSCs迁移的影响。采用酶联免疫吸附测定(ELISA)法检测放射性迁移系统及Transwell培养上清液中SDF-1及血管内皮生长因子(vascular endothelial growth factor,VEGF)的含量。结果:经过不同时间点的观察,放射性迁移、划痕修复动态检测以及Transwell趋化性迁移均可见不同程度的NSCs的迁移;与空白组比较,补阳还五汤及川芎嗪能够明显促进放射性迁移体系中NSCs的迁移,还能够显著增加向Transwell下室迁移的细胞数,且具有剂量依赖性(P<0.05,P<0.01),此外还可显著提高NSCs培养上清中SDF-1,VEGF的含量(P<0.01),其中对SDF-1含量上调作用高于VEGF;给予AMD3100预处理后,600 mg·L~(-1)补阳还五汤及10,50 mg·L~(-1)川芎嗪促进Transwell向下室迁移的细胞数显著降低。结论:该研究建立的NSCs迁移研究平台动态、多维,能模拟不同临床病理生理过程,且经济实用、简便易行,可供中药复方和单体成分活性筛选之用。
OBJECTIVE: To establish a platform for the study of migration of neural stem cells (NSCs) in vitro and to verify the suitability of this tablet with classic nourishing yang huang hua wu decoction. Methods: NSCs were isolated and cultured in vitro. The NSCs migration research platform was established by using radioactive migration of NSCs, dynamic detection of scratch repair and detection of chemotactic migration by Transwell. The platform was used to evaluate the effects of classic prescriptions such as Buyang Huanwu Decoction (300,600 mg · L -1), tetramethylpyrazine (10,50 mg · L -1) and positive control stromal cell derived factor - 1 (stromal-derived factor 1, SDF-1) on the migration of NSCs. The content of SDF-1 and vascular endothelial growth factor (VEGF) in radioactive migration system and Transwell culture supernatant were detected by enzyme-linked immunosorbent assay (ELISA). Results: After different time points, the migration of NSCs was observed by dynamic detection of radioactive migration, scratch repair and chemotactic migration of Transwell. Compared with the blank group, Buyang Huanwu Decoction and ligustrazine could significantly promote the radiation migration The migration of NSCs in the system also significantly increased the number of cells migrating to the lower compartment of Transwell in a dose-dependent manner (P <0.05, P <0.01). In addition, the migration of NSCs in culture supernatants significantly increased the expression of SDF-1 and VEGF (P <0.01). The up-regulation of SDF-1 was higher than that of VEGF. After pretreatment with AMD3100, 600 mg · L -1 BUYH decoction and 10, 50 mg · L -1 ) Tetramethylpyrazine significantly reduced the number of cells translocating to the lower chamber. Conclusion: The research platform of NSCs migration established in this study is dynamic and multidimensional and can simulate different clinicopathological pathophysiological processes. It is economical and practical, simple and easy to use, and can be used for the activity screening of traditional Chinese medicine compounds and monomer components.