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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 effect of overexpression of Mash-1 gene on the differentiation of mouse embryonic stem cells (ESCs) into neurons. Methods MSCV-Mash-1 gene and MSCV empty vector were infected into mouse ESCs (MSC3-MSC-Mash-1 group, MSCV-CE3 group) by virus infection and Mash-1 gene was identified by RT- Then, the embryoid body was formed by the hanging drop culture method and then induced to differentiate by neural culture. The morphological changes of the cells were observed under an inverted phase contrast microscope at 7 and 21 days. The positive expression of nestin and β-tubulin Ⅲ in neural stem cells and neuronal markers were detected by immunofluorescence staining. The expression of AFP (endoderm marker), Brachyury (mesoderm) and FGF-6 were detected by real-time fluorescence quantitative PCR at 0,1,7,14,21 days. 5 (ectoderm marker gene), Oct3 / 4 (ESC marker gene), nestin (neural stem cell marker gene) and β-tubulinⅢ (neuron marker gene) expression. Normal CE3 cells served as control (CE3 group). Results Compared with MSCV-CE3 group and CE3 group, the expression of Mash-1 gene in MSCVMash-1-CE3 group was significantly increased. After cultured for 7 and 21 days, the cells in MSCV-Mash-1-CE3 group showed enhanced refraction, elongated slender axons, monopolar, bipolar or multipolar morphology, showing neural stem cells and nerves Cell morphology; MSCV-CE3 group and CE3 group only observed a few cells grow elongated axons. Immunofluorescence staining showed that the positive rate of nestin in 7 days and the positive rate of 21 dβ-tubulinⅢ in MSCV-Mash-1-CE3 group were significantly higher than that in MSCV-CE3 group and CE3 group (P <0.05). Real-time quantitative PCR showed that the expression of Brachyury in MSCV-Mash-1-CE3 group was significantly lower than that in CE3 group and MSCV-CE3 group (P <0.05), while the expression of FGF-5 and nestin increased ). The expression of β-tubulinⅢ also increased significantly after 7 days (P <0.05). There was no significant difference between the CE3 group and MSCV-CE3 group (P> 0.05). There was no significant difference in the expression of AFP and Oct3 / 4 between the three groups at all time points (P> 0.05). Conclusion Overexpression of Mash-1 gene can promote the differentiation of mouse ESCs into neurons.