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

Objectives: 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).

Conclusions: 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.