中文摘要：

目的构建亚甲基四氢叶酸还原酶（MTHFR）特异性小干扰RNA（siRNA）真核表达载体，体外观察其对MTHFR基因的沉默作用。方法采用基因克隆技术，将合成的特异性MTHFR RNA干扰寡核苷酸序列插入真核表达载体PsiRNA—Hh1neoG2，构建MTHFR siRNA真核表达载体。应用核转染技术将PsiRNA—MTHFR重组质粒分别导入原代培养的小鼠胚胎腭突间充质（MEPM）细胞。48h和5d后用实时定量聚合酶链反应（Real—TimePCR）和Western blot技术检测MEPM细胞内MTHFR mRNA及蛋白水平的表达情况。结果成功构建PsiRNA—MTHFR真核表达载体。经检测48h和5d后转染PsiRNA—MTHFR的MEPM细胞MTHFR mRNA及蛋白表达均显著下调。结论构建的RNA干扰真核表达载体能明显干扰MTHFR mRNA及蛋白的表达，为进一步研究MTHFR的功能以及其调控胚胎腭突融合的机制奠定基础。

英文摘要：

Objective To construct a small interfering RNA （siRNA） enkaryotic expression vector specific for methylene tetrahydrofolate reduetase （MTHFR） gene and to observe its silencing effect on MTHFR gene. Methods The expression vectors of PsiRNA-MTHFR were constructed by gene recombination and then were nucleofected into the primary cultured MEPM cell. At 48 h and 5 d after nucleofection, the expression of MTHFR in the levels of mRNA and protein was detected by real-time quantitative polymerase chain reaction （Real-Time PCR） and Western blot. Results The eukaryotic expression vector of PsiRNA-MTHFR, which significantly down-regulated mRNA and protein of MTHFR at 48 h and 5 d after nucleofection, were successfully constructed. Conclusion Eukaryotic expression vector of siRNA specific for MTHFR is successfully contructed, which lays the basis for its application in the mechanism research of MTHFR gene regulating embryo palate shelves fusion.