Expression Of Satb2 And Hoxa2 During The Formation Of Cleft Palate Induced By Retinoic Acid

[Background] Congenital cleft palate(CP) is a common developmental deformity of oral and maxillofacial region, considered to be a multi-gene genetic disease currently, which is caused by the combined effect of genetic and environmental factors, its pathogenesis may be related to abnormal proliferation and apoptosis of cell,abnormal expression of many cytokines and their receptors, but the exact molecular mechanism of cleft palate has not been fully elucidated. Studies show that in embryonic development, retinoic acid plays an important regulatory role in organ formation,cell proliferation and differentiation by interaction with homeobox(hox) gene family, but excessive retinoic acid may lead to congenital cleft palate occurs, may be related to abnormal proliferation,apoptosis of embryonic palate mesenchymal cell (EPMC) and abnormal expression of the related gene in palatal development, clinical studies have found homeobox gene Satb2 and Hoxa2 mutation also closely related to cleft palate. Therefore, we establish a mouse model of cleft palate by all-trans retinoic acid (atRA), to detect proliferation of EPMC and expression of Satb2,Hoxa2 during palatal development,to investigate the effect of Satb2 and Hoxa2 on the formation of cleft palate induced by all-trans retinoic acid, to provide theoretical and experimental basis for further revealing the molecular mechanism of cleft palate and designing related control measures .[Objective] To establish all-trans retinoic acid induced C57BL / 6 mouse model of cleft palate, detect change characteristics of Satb2,Hoxa2 expression during palatal development and cleft palate, explore the relevant mechanisms.[Methods] AtRA and corn oil was intragastric administered as experimental group while only corn oil was intragastric administered as control group on gestation day (GD)11.5 ,the pregnant mice were sacrificed and fetal mice were dissected at GD14.5, GD18.5 respectively, morphology and structure of fetal mice palate were observed by stereology and histological staining;5-bromo-2-deoxyuridine (BrdU) was intraperitoneally injected at 1.5 hours before killing, EPMC proliferation of two group was detected at two different time points;Satb2 and Hoxa2 expression of mRNA and protein levels in palatal process were detected by Real-time quantitative PCR, Western blot and immunohistochemical staining.[Results] 1. Stereologic observation and histological staining show in the experimental group, 35 fetuses manifest cleft palate in total 37 fetuses at GD18.5, incidence of cleft palate is 95%, in the control group, no fetal malformation occur in total 38 fetuses at GD18.5. 2. BrdU labeled proliferation show the EPMC proliferation is lower in the experimental group at GD14.5 in comparison with the control group, there is a significant difference between two groups(P<0.05),BrdU labeled positive cells decrease significantly in the experimental group at GD18.5 in comparison with the control group, due to the uneven distribution of positive cells, statistically analysis is impossible between two groups. 3. Real-time quantitative PCR results show that Hoxa2 mRNA expression at GD14.5 in the experimental group were significantly higher than that of control group, there is a significant difference between two groups(P<0.05), Hoxa2 mRNA expression at GD18.5 is no significant difference between two groups(P>0.05);Satb2 mRNA expression is no significant difference at GD14.5 or GD18.5 between experimental and control groups (P>0.05);Hoxa2 mRNA expression is decreased at GD18.5 in the experimental group and control group than that of GD14.5(P<0.05), Satb2 mRNA expression is increased at GD18.5 in the two groups than that of GD14.5(P<0.05) . 4. Western blot show Hoxa2 expression is increased in the experimental group at GD14.5 and GD18.5;immunohistochemical staining show Satb2,Hoxa2 expression is no notable change in the experimental group and control group, Hoxa2 express strongly in the palatal mesenchyme and Satb2 express strongly in the epithelium.[Conclusion] The reliable mouse model of cleft palate can be established with atRA ; atRA inhibit EPMC proliferation and differentiation probably through promotion Hoxa2 expression in the early phase of palatal development, thus affect the normal developmental patterns of the palate, result in cleft palate finally.