RNA干扰原代培养胚胎腭间充质细胞Smad2/3基因对atRA阻滞细胞周期作用的影响

目的：证实atRA可以通过Smad2/3作用于p21,从而导致胚胎腭间充质细胞周期受阻而出现腭裂。方法：原代培养胎鼠胚胎腭间充质细胞并进行鉴定。采用RNA干扰原代培养胚胎腭间充质细胞Smad2/3基因后,Western印迹检测Smad2/3、pSmad2、pSmad3和p21的蛋白表达水平及干扰后细胞周期的变化。采用SPSS11.0软件包对数据进行单因素方差分析和t检验。结果：经过免疫组化鉴定,证实原代培养C57BL/6N胎鼠腭突间充质细胞成功,并且Smad2/3siRNA可以有效干扰Smad2/3在原代培养MEPM细胞中的表达。Western印迹检测结果证实,Smad2/3siRNA可以通过敲减atRA诱导的Smad2/3表达增高现象,继而降低atRA诱导的p21表达增高现象。此外,Smad2/3siRNA在一定程度上降低了atRA诱导的胎鼠腭突间充质细胞G1期阻滞现象。结论：Smad2/3通过p21参与atRA诱导的胚胎腭突间充质细胞G1期阻滞现象。

The effect of smad 2/3 RNA interference on growth inhibition of mouse embryonic palatal mesenchymal cells induced by all-trams retinoic acid

PURPOSE：To elucidate the mechanism by which all-trans retinoic acid（atRA） induces the cleft palate.METHODS：Primary cultured embryonic palatal mesenchymal cells were used in the study.The mouse Smad2/3 siRNA was applied to knockdown Smad2/3 expression of primary cultured embryonic palatal mesenchymal cells.The efficiency of target gene knockdown was verified by Western blotting.The cell cycle distribution in mouse embryonic palatal mesenchymal cells was detected with flow cytometry after Smad2/3 RNAi.The data were analyzed with SPSS 11.5 software package for one-way ANOVA and Student＇s t test.RESULTS：The embryonic palatal mesenchymal cells were successfully cultured.However,p21 expression levels were decreased in the Smad2/3 siRNA transfected group than that in the control siRNA transfected group through Western blotting analysis.G0/G1 arrest was partially released following Smad2/3 siRNA knockdown.CONCLUSIONS：Our study demonstrates that Smad2/3 regulation of p21 was partly required for atRA-induced cell cycle perturbations in MEPM cells.