过表达Mash-1-1基因促进小鼠胚胎干细胞向神经细胞分化的研究

发布时间：2019-02-13 00:32

【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(P0.05)。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(P0.05),FGF-5和nestin表达增高(P0.05);7 d后β-tubulinⅢ表达亦显著增高(P0.05);CE3组及MSCV-CE3组以上指标比较,差异均无统计学意义(P0.05)。3组间各时间点AFP和Oct3/4表达比较,差异均无统计学意义(P0.05)。结论过表达Mash-1基因能促进小鼠ESC向神经细胞方向分化。
[Abstract]:Objective to investigate the effect of overexpression of Mash-1 gene on the differentiation of mouse embryonic stem cells (embryonic stem cells,ESC) into neural cells. Methods MSCV-Mash-1 gene and MSCV empty vector were infected into ESC (CE3 cells) of mice (MSCV-Mash-1-CE3 group, MSCV-CE3 group) by virus infection technique. RT-PCR was used to identify the expression of Mash-1 gene. Then suspension culture method was used to induce the formation of embryoid body, and then differentiation was induced by adherent neural media. The morphological changes of the cells were observed under inverted phase-contrast microscope for 21 days, and the positive rates of nestin (nestin) and 尾 -tubulin 鈪，

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