The Effect And Mechanism Of Over Doses Atra On Cleft Palate Formation

Cleft palate is one of common birth defects and occurs in about one in1000live births in China. The palate formation is a complex process involving multiple events, including palatal shelf growth, elevation, and fusion. Cleft palate might be caused by the failure of palatal midline epithelial seam to degenerate, which is essential for palatal mesenchymal confluence. Therefore, the normal fate of medial edge epithelium cells plays a key role in palatal confluence. All-trans retinoic acid (atRA) is one of the oxidative metabolites from vitamin A. It is essential for the development of mammalian embryos. However, in excess or deficiency, it is teratogenic for both animals and humans, such as cleft palate and neural tube defects. Moreover, atRA controls the expression of TGF-β in a variety of systems. Palate formation is Smad-dependent TGF-β signaling pathway. Whether atRA could affect the expression of Smad in palate is unknown.ObjectivesCell scratch assay was carried out to observe the influence of excessive atRA on migration of medial edge epithelium (MEE) cells. Histology, immunocytpochemistry, TUNEL, as well as Western blot methods were applied to investigate the effects of high doses atRA on palatal fusion, apoptosis of MEE cells, and the expressions of the basal laminin and Smad.MethodsKunming mice at10-weeks of age were mated at the ratio of1:2(male:female). The presence of vaginal plug was defined as the gestation day (GDO). On GD13, the dams were killed and the fetuses were dissected out and their palate shelves were isolated. Dispase was used to separate MEE cells from the underlying basement membrane. These primary MEE cells were inoculated at the density of5×105/ml in25ml culture flask, which containing5ml of DMEM/F-12medium in a cell culture chamber for96h. Cell layer scratching experiments were performed in a6-well-plate under the same cultivation condition and with same cell density, of which, three wells belonged to control group and3belonged to atRA group, the culture medium of the latter contained5μmol/L of atRA. When the cultured epithelial cell covered80%of the plate well, a scratching line paralleled with the long axis of plate was made by using a20μl pipetting tip to observe the influence of atRA on MEE cell migration.Twelve of24pairs of isolated palatal shelves were separately placed in Grobstein organ petri dish which contained0μmol/L,1μmol/L,5μmol/L,10μmol/L of atRA, and followed by72h incubation in the cell culture chamber. Then, all of the palatal shelves were fixed by formalin and imbedded with paraffin, then cut into slices at5μm. The slices from each group were stained with hematoxylin-cosin to investigate the effect of atRA on palatal fusion at different doses. The cutting slices from both5μmol/L atRA of culture medium dose and control group were checked by the TUNEL method, then the same sections were carried out by immunofluorescence procedure for observation under a fluorescence microscope. The center part1mm strips of the remaining palatal shelves which were previously treated with0μmol/L and5μmol/L of atRA were harvested after24h incubation and the Smad expression was measured via Western blot method.ResultsAfter cultivation, primary MEE cells showed normal appearence, cobble-shaped and rich in cellular plasma. When more than80%of the MEE cells fused together, they looked like an arrangement of slabstone. During the cultivation, MEE cells in the control group grew toward the scratching line from the sides of scratch and almost covered the whole scratch area by the end of culture period. In contrast, no migration growth was seen in the atRA treated groups MEE cells.The control group palates fused completely, the shelves became continuous, while, palatal shelves treated with atRA at1μmol/L and5μmol/L still had medial edge epithelium seam. For the palate treated with atRA at10μmol/L atRA showed no contact in the midline.In control palates, a mesenchymal confluence was established across the palate, the basal laminin within the basement membrane was disintegrated and showed large amount of apoptotic cells in the epithelial triangles of mouth and nasal cavity; In the 5μmol/L atRA-treated groups, TUNEL test showed almost no cell apoptosis in MEE cells, and the basement membrane was mostly intact. Western blot assay demonstrated that atRA treated palate down-regulated phosphorylation of Smad2and Smad3, but up-regulated the expression of Smad7.ConlusionsMEE cell migration and apoptosis are the two important aspects during palate fusion. The disruption of any of the aspects might cause cleft palate.In the palatal development, excessive atRA could inhibit both cell migration and apoptosis of the MEE cell, and prevent the basal laminin disintegration, which is associated with cleft palate formation.Abnormal apoptosis of palatal MEE cells induced by excessive atRA might be related to the alteration of protein expression for Smad family.