摘要
目的探讨过表达Mash-1基因对小鼠胚胎干细胞(embryonic stem cells,ESC)向神经细胞分化的影响。方法采用病毒感染技术将MSCV-Mash-1基因、MSCV空载体感染至小鼠ESC(CE3细胞)(MSCV-Mash-1-CE3组、MSCV-CE3组),RT-PCR鉴定细胞Mash-1基因的表达情况;然后采用悬滴培养法使其形成拟胚体,再经神经培养液贴壁诱导分化。培养7、21 d于倒置相差显微镜下观察细胞形态改变;免疫荧光染色检测细胞贴壁后其神经干细胞和神经元标记蛋白巢蛋白(nestin)和β-微管蛋白Ⅲ(β-tubulinⅢ)的阳性率;实时荧光定量PCR检测诱导培养0、1、7、14、21 d时,细胞甲胎蛋白(α-fetal protein,AFP)(内胚层标记基因)、Brachyury(中胚层标记基因)、FGF-5(外胚层标记基因)、Oct3/4(ESC标记基因)、nestin(神经干细胞标记基因)和β-tubulinⅢ(神经元标记基因)表达情况。以正常CE3细胞作为对照(CE3组)。结果与MSCV-CE3组和CE3组比较,MSCVMash-1-CE3组细胞Mash-1基因表达明显升高。经神经培养液诱导培养7、21 d后,MSCV-Mash-1-CE3组可见许多细胞折光性增强,长出细长轴突,出现单极、双极或多极形态,呈神经干细胞和神经元细胞形态;MSCV-CE3组和CE3组仅能观察到少数细胞长出细长轴突。免疫荧光染色检测示MSCV-Mash-1-CE3组诱导分化7 d nestin阳性率和21 dβ-tubulinⅢ阳性率显著高于MSCV-CE3组及CE3组(P<0.05)。实时荧光定量PCR检测示,与CE3组及MSCV-CE3组比较,培养1 d后MSCV-Mash-1-CE3组Brachyury表达明显降低(P<0.05),FGF-5和nestin表达增高(P<0.05);7 d后β-tubulinⅢ表达亦显著增高(P<0.05);CE3组及MSCV-CE3组以上指标比较,差异均无统计学意义(P>0.05)。3组间各时间点AFP和Oct3/4表达比较,差异均无统计学意义(P>0.05)。结论过表达Mash-1基因能促进小鼠ESC向神经细胞方向分化。
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-I-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-I-CE3 group was significantly increased. At 7 and 21 days after neural induction cultured, cells in MSCV-Mash-I-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 MSCV-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.
出处
《中国修复重建外科杂志》
CAS
CSCD
北大核心
2015年第12期1553-1559,共7页
Chinese Journal of Reparative and Reconstructive Surgery
