MTHFR基因沉默对小鼠胚胎腭突间充质细胞增殖和凋亡的影响

MTHFR gene silencing affects mouse embryonic palatal mesenchymal cell proliferation and apoptosis

目的:应用RNA干扰技术,从细胞水平研究5,10-亚甲基四氢叶酸还原酶(MTHFR)基因突变导致腭裂发生的机制。方法:构建MTHFR基因沉默载体,对13天胚胎的腭突间充质细胞进行RNA干扰实验。MTT法检测MTHFR基因沉默后细胞增殖的改变,流式细胞仪检测MTHFR基因沉默后对胚胎腭突间充质细胞凋亡的影响。实验结果采用SPSS11.0软件包进行分析,细胞增殖的比较用重复测量方差分析,凋亡率检测应用U检验。结果:MTHFR基因表达下调后,在无叶酸存在的情况下,腭突间充质细胞的增殖速度明显减慢,与对照组相比有显著性差异(P<0.001);实验组的凋亡率显著高于对照组(P<0.05)。结论:在外源性叶酸补充不足的情况下,腭突间充质细胞增殖减弱并发生凋亡,这可能是MTHFR基因突变与先天性唇腭裂发生相关的病理学基础。

PURPOSE： RNA interferenee （RNAi） was applied to knock down MTHFR in mouse embryonie palatal mesenehymal （EPM） cells,in order to understand the role of MTHFR gene variants in pathogenesis of non-syndromic eleft lip and palate （NCLP）. METHODS： The primary cultures of EPM cells were transfected into construction of MTHFR ShRNA expression vector, the effects of MTHFR gene silencing on the proliferation of MEPM cells were measured with an MTT assay and cell apoptosis assay by FCM. The results of the experiment were analyzed using SPSS software package. ANOVA was used to analyze the cell proliferation and U test was used to deteet the apoptosis rate. RESULTS： The results showed the cells of MTHFR gene silencing grew more slowly than the cells of control group. MTHFR gene silencing resulted in significant reduction in cell proliferation rate （P〈0.001） and a significant increase in apoptotie eell number （P〈0.05）. CONCLUSION： The results indicate that MTHFR gene silencing effect EPM cell proliferation and cell apoptosis in the state of both MTHFR gene variants and insufficient folie acid supplement. It may be the pathologic basis for the correlation between MTHFR gene mutation and NCLP. Supported by National Natural Seience Foundation of China （Grant No.30371552）.