[OVER EXPRESSION OF Mash-1 GENE IN PROMOTING DIFFERENTIATION OF MOUSE EMBRYONIC STEM CELLS INTO NEURAL CELLS].

Objective: To investigate the effects of over expression of Mash-1 gene on the differentiation of embryonic stem cells (ESC) into neural cells in vitro.

Methods: The ESC of rats (CE3 cells) were transfected with MSCV-Mash-1 (MSCV-Mash-1-CE3 group) or MSCV (MSCV-CE3 group). The expression of Mash-1 gene was detected by RT-PCR. After transfection, hanging-drop culture was used to form embryonic bodies, and then embryonic bodies were cultured with neural induction medium. The cell morphology was observed under inverted phase contrast microscopy at 7 and 21 days; the positive rates of neural stem cells marker protein (nestin) and neuron marker protein (β-tubulin III) were measured by immunofluorescence staining after cell attachment; and the gene expressions of a-fetal protein (AFP), Brachyury, fibroblast growth factor 5 (FGF-5), Oct3/4, nestin, and β-tubulin III were detected by real-time fluorescence quantitative PCR at 0, 1, 7, 14, and 21 days after culture. The CE3 cells were used as control (CE3 group).

Results: Compared with MSCV-CE3 and CE3 groups, the expression of Mash-1 gene in MSCV-Mash-1-CE3 group was significantly increased. At 7 and 21 days after neural induction cultured, cells in MSCV-Mash-1-CE3 group had axons growth and showed neural stem cell-like and neuron cell-like morphology (unipolar, bipolar, and multipolar neurons), but few cells had axons growth in MSCV-CE3 and CE3 groups. The positive rates of nestin at 7 days and β-tubulin III at 21 days in MSCV-Mash-1-CE3 group were significantly higher than those in MSCV-CE3 and CE3 groups (P < 0.05). Real-time fluorescence quatitative PCR results showed that the gene expression of Brachyury was significantly decreased after 1 day (P < 0.05), and the gene expressions of FGF-5 and nestin were significantly increased after 1 day (P < 0.05) in MSC V-Mash- 1-CE3 group when compared with CE3 and MSCV-CE3 groups; the gene expression of β-tubulin III was significantly increased after 7 days (P < 0.05). There was no significant difference in above indexes between CE3 and MSCV-CE3 groups (P > 0.05). The expressions of AFP and Oct3/4 showed no significant difference among groups at each time point (P>0.05).

Conclusion: Over expression of Mash-1 gene can promote differentiation of ESC into neural cells in vitro.