川芎嗪对体外培养的胎鼠神经干细胞增殖和分化的影响

目的：探讨川芎嗪（TMP）在体外神经干细胞（NSCs）增殖与分化中的作用。方法：原代提取孕14 d雌性大鼠的胎鼠大脑皮层分离培养，并作免疫荧光染色鉴定，取传代培养第3代的NSCs进行实验。实验分为对照组、β-巯基乙醇阳性对照组、TMP诱导组和TMP+EGTA组（n=4）。采用BrdU法和MTT法观察川芎嗪对NSCs增殖数量的影响，采用蛋白免疫印迹法检测NSCs的分化表达情况。结果：实验成功分离纯化原代NSCs，培养3~5 d可见部分神经球形成，具备典型的NSCs形态并表达NSCs特异抗原巢蛋白；BrdU法和MTT法结果均显示，与对照组和β-巯基乙醇阳性对照组相比，TMP组NSCs增殖数量明显增多（P<0.05）；蛋白免疫印迹结果显示，TMP组和TMP+EGTA组NSCs的神经元分化率明显增高，TMP+EGTA组分化率增高更明显（P<0.05）。结论：TMP能显著增强NSCs的增殖和神经元分化率。减少细胞外Ca²⁺可促进TMP诱导NSCs向神经元分化，Ca²⁺信号在TMP诱导NSCs向神经元分化过程中起重要作用。

Effects of tetramethylypyrazine nitrone on proliferation and differentiation of neural stem cells in vitro

Objective:To investigate the role of tetramethylpyrazine(TMP) nitrone in proliferation and differentiation of neural stem cells (NSCs). Methods:We separated and cultivated the original generation of NSCs from cerebral cortex of 14 days rat embryo, and the phenotype characteristics of the third-generation NSCs was tested by immunofluorescence. The experiment was divided into control group, β-mercaptoethanol positive control group, tetramethylpyrazine nitrone group and tetramethylpyrazine nitrone + ethylene glycol tetraacetic acid(EGTA) group (n=4). The third-generation cultivation of NSCs was used in the experiment. The effect of tetramethylpyrazine nitrone on the number of NSCs proliferation was determined by BrdU and MTT, and the differentiation of NSCs was determined by Western blot. Results:The primary NSCs was isolated successfully, neurospheres with typical NSCs morphology and expressing nestin was formed at 3-5 days. As BrdU and MTT assay results shown, compared with the control group andβ-mercaptoethanol positive control group, the NSCs proliferation numbers of tetramethylpyrazine nitrone group increased significantly(P<0.05). The results of Western blot showed that the neuronal differentiation rate of NSCs was increased significantly in both the tetramethylpyrazine nitrone group and tetramethylpyrazine nitrone + EGTA group, and the differentiation rate of NSCs in tetramethylpyrazine nitrone + EGTA group increased more significantly(P<0.05). Conclusion:Tetramethylpyrazine nitrone can significantly enhance the proliferation and neuronal differentiation rate of NSCs. Decrease in extracellular Ca²⁺ can promote the differentiation of NSCs into neurons induced by tetramethylpyrazine nitrone. Ca²⁺ signaling plays an important role in the differentiation of NSCs into neurons.