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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。
OBJECTIVE: To demonstrate that atRA can act on p21 via Smad2 / 3, resulting in cleft palate resulting from the blocked cycle of embryonic mesenchymal cells. Methods: The fetal rat embryonic palatal mesenchymal cells were primary cultured and identified. After Smad2 / 3 gene was primary cultured by RNA interference, the expression of Smad2 / 3, pSmad2, pSmad3 and p21 protein and cell cycle were detected by Western blotting. SPSS 11.0 software package was used to analyze the data by one-way ANOVA and t-test. Results: After immunohistochemical identification, it was confirmed that primary culture of fetal rat palatal mesenchymal cells of C57BL / 6N was successful, and Smad2 / 3 siRNA could effectively interfere the expression of Smad2 / 3 in primary cultured MEPM cells. Western blot results confirmed that Smad2 / 3 siRNA knocked down atRA induced Smad2 / 3 expression increased, and then reduced atRA induced p21 expression increased. In addition, Smad2 / 3siRNA decreased the atRA induced G1 phase arrest of fetal rat palatal mesenchymal cells to a certain extent. CONCLUSIONS: Smad2 / 3 is involved in atRA induced G1 arrest in embryonic palate mesenchymal cells via p21.