神经干细胞迁移研究平台的建立及补阳还五汤的干预影响

Establishment of Platform for Neural Stem Cells Migration in Vitro and Effect of Buyang Huanwu Tang on Migration

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 migration platform for neural stem cells（ NSCs） in vitro and verify the applicability of this platform by using Buyang Huanwu Tang,a Chinese classical formula. Method： NSCs were isolated from rat embryos,and used in establishing a migration platform for neural stem cells in vitro by radial cell migration assay,scratch repair dynamic detection,and Transwell chemotaxis system. Then this platform was used to evaluate the effects ofof Buyang Huanwu Tang（ 300,600 mg·L^-1）,Tetramethylpyrazine（ 10,50 mg·L^-1）,as well as stromal-derived factor 1（ SDF-1） on NSCs migration. ELISA assay was used to detect the contents of migration regulatory factor SDF-1 and vascular endothelial growth factor（ VEGF） in the radial cell migration and transwell system. Result： NSCs migration in varying degrees was observed at different time points in radial migration,scratch repair dynamic detection and transwell chemotaxis system. As compared with the blank group,Buyang Huanwu Tang and Tetramethylpyrazine could significantly promote NSCs migration in radio migration system,but also significantly increase the number of NSCs migrated to lower chamber in Transwell system（ P〈0. 05,P〈0. 01） in a dose-dependent manner. Additionally,Buyang Huanwu Tang and Tetramethylpyrazine could promote the expression levels of SDF-1 and VEGF in NSCs culture supernatant（ P〈0. 01）,where the upregulation effect on SDF-1 was higher than that on VEGF. We also found that the effect of Buyang Huanwu Tang（ 600 mg·L^-1） and Tetramethylpyrazine（ 10,50 mg·L^-1） were inhibited after AMD3100 pretreatment,where the number of NSCs migrated to lower champ was reduced significantly. Conclusion： The NSCs migration research platform established in this study is dynamic and multi-dimensional. It can simulate different clinical pathophysiological processes,and in addition,this platform is economical,practical and easy to operate.