益气活血化痰中药复方对大鼠胚胎神经干细胞生长分化增殖的影响机制研究

Effects of Yiqi Huoxue Huatan Formula on proliferation and differentiation of rat embryonic neural stem cells

目的研究益气活血化痰药物血清对体外培养的大鼠胚胎神经干细胞(NSC)生长分化增殖的影响。方法细胞分为空白血清组、含药血清组、阻滞剂组、含药血清+阻滞剂组和培养基对照组5组。采用细胞培养、血清药理学、免疫荧光等技术,观察中药复方血清对大鼠NSC生长分化的影响;相差显微镜进行形态学观察;免疫荧光染色技术,检测各组细胞nestin、神经元特异性烯醇化酶(NES)和胶质纤维酸性蛋白(GFAP)的表达,观察神经干细胞的分化。结果益气活血化痰中药对神经干细胞无毒性,并能够促进神经干细胞生长分化。nestin蛋白在诱导分化24 h内表达最强,诱导72 h后nestin表达明显减弱直至消失。益气活血化痰中药组NES和GFAP阳性细胞数明显增加。结论益气活血化痰药物血清有促进NSC生长分化增殖的作用。

Objective To study the effects of Yiqi Huoxue Huatan（ qi-replenishing blood-activating phlegm-resolving） Formula on proliferation and differentiation of rat embryonic neural stem cells（ NSC）.Methods The rat NSC were divided into five groups,including blank serum group,drug serum group,blank serum ＋ LY294002 group,drug serum ＋ LY294002 group,and culture medium control group.The effects of drug serum on cell proliferation and differentiation were evaluated by cell culturing,serum pharmacology,immunofluorescence techniques. Morphological changes were observed by phase contrast microscope. Nestin,neuron-specific enolase（ NES） and glial fibrillary acidic protein（ GFAP） were measured by using immunofluorescence staining. Results Morphologically,drug serum was non-toxic to neural stem cells and promoted the differentiation of neural stem cells. Immunofluorescence staining showed that nestin expression increased within 24 h yet decreased significantly after 72 h incubation. NES and GFAP positive cells increased significantly in the drug serum group,compared with control group.Conclusion The serum from the rats treated with Yiqi Huoxue Huatan Formula has significant effects on differentiation and proliferationof NSC.