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目的探讨茶氨酸对神经干细胞增殖和分化的影响。方法采用机械吹打结合200目钢网滤过的方法,从12.5 d的昆明胎鼠大脑中分离NSCs并培养;用巢蛋白(nestin)免疫荧光染色鉴定NSC;加入不同浓度茶氨酸(theanine),MTS法检测不同时间点活性,选定茶氨酸最终加药浓度(100μmol/L);将NSCs分为两组:对照组、加药组(茶氨酸浓度为100μmol/L);培养后进行Brd U染色和微管相关蛋白2(MAP2)、胶质纤维酸性蛋白(GFAP)免疫荧光染色;利用Q-PCR以及Westen blotting检测MAP2和GFAP表达。结果 100μmol/L茶氨酸处理组M TS检测的吸光度值显著增高(P<0.05);加药组Brd U阳性细胞比率、M AP2阳性细胞比率明显高于对照组(P<0.05),两组GFAP阳性细胞比率无统计学差异;加药组MAP2表达明显高于对照组(P<0.05),两组GFAP表达无统计学差异。结论茶氨酸可促进NSCs增殖及其向神经元方向分化。
Objective To investigate the effects of theanine on the proliferation and differentiation of neural stem cells. Methods NSCs were isolated from the brains of Kunming fetus at 12.5 days by mechanical blowing and 200 mesh stencil filtration. NSCs were identified by nestin immunofluorescence staining. Theanine was added at different concentrations, MTS method was used to detect the activities at different time points. The final dosing concentration of theanine (100μmol / L) was selected. NSCs were divided into two groups: control group and dosing group (theanine concentration was 100μmol / L) BrdU staining, microtubule-associated protein 2 (MAP2) and glial fibrillary acidic protein (GFAP) immunofluorescence staining. The expression of MAP2 and GFAP was detected by Q-PCR and Westen blotting. Results The absorbance value of M TS in 100 μmol / L Theanine treatment group was significantly increased (P <0.05). The BrdU positive cells ratio and M AP2 positive cells ratio in the dosing group were significantly higher than those in the control group (P <0.05) There was no significant difference in the number of GFAP positive cells between the two groups. The expression of MAP2 in the dosing group was significantly higher than that in the control group (P <0.05). There was no significant difference in GFAP expression between the two groups. Conclusion Theanine can promote the proliferation of NSCs and their differentiation into neurons.