Study On The Expression Of TGF-β/Smad2/3in Retinoic Acid-induced Cleft Palate In Mice

Cleft palate (cleft palate, CP) is one of the most common congenital malformation, regulating by both multiple interacting genetic and environmental factors in the earlier period of embryos. The rate of occurrence has up to about1.82%o in China, which brings huge suffering to patients, their families and society. It is so complicated that the mechanism of cleft palate remains not fully elucidated. Therefore, the study on the etiology of the CP have still considerable importance in teratologic research.The atRA, a metabolite of vitamin A, is an essential morphogen during embryonic development. However, it was proved that excess of the atRA has distinct teratogenic action compared to other drugs. The atRA is closely related with TGF-β, and it can regulate the expression of TGF-β on palatal shelves fusion. And the atRA and TGF-β signaling pathways are very important for cellular proliferation and differentiation. Presently, Smads is identified as the only substrates for TGF-β receptor, and TGF-β can be achived by Smad protein in cytoplasm. Smad2/3can be phosphorylated by TGF-β receptors.And then transfer to the nucleus with TGF-P signal, where they regulate the expression of the target gene. There are a number of experimental evidence which indicate that TGF-β regulates these processes in the development of palate shelves. However, although there is basic evidence of palate between them in vitro, the relationship between atRA and Smad signaling pathways, is poorly understood.This paper aims to establish mice models for CP induced by excess atRA in C57BL/6J mouse,and to observe the expression and distribution changes of Smad2/3during the period of the development of CP induced by excess atRA with the techniques of stains and immunohistochemistry. Therefore, the present study can provide scientific basis for the prevention and treatment of CP.Material and Methods1.Establishing CP model induced by atRA:we elect C57BL/6J mouse about10weeks of age, weighting20～25g. Housed overninght at8pm F:M=2:1, checked for vaginal plugs the next morning8am,which was considered gestation day0.54Pregnant mice were randomly devided into atRA-treated group, vegetable oil control group and blank control group, and each group have18mice. The atRA-treated group were orally treated with a single dose of10mg/ml atRA vegetable oil solution per O.Olml/g on GD10. The vegetable oil control group were administrated with a volume of vegetable oil, equal to the highest dose of atRA, and blank control group do not deal with anything. All animals were killed on GD1318, GD148, GD1418, GD158, GD1518, GD168respectively, and take out cyema.Cyema’s head be fixed,bladed after embed. Microscopic changes in CP were evaluated in the specimens, sectioned routinely in5μm thickness at a transverse plane with the paraffin microtome and stained with HE. The embryonic palatal development were examined by stereo microscope and light microscopy.2. Detection of Smad2/3:Through in immunohistochemistry, the difference of Smad2/3expression between normal and defected cleft palate induced by atRA was analyzed in mouse. Through in immunohistochemistry, the difference of Smad2/3expression between normal and defected cleft palate induced by atRA was analyzed in mouse.Results1. The effect of atRA on the CP:Small palate shelves developed in atRA group,and they were delayed both the lift of palate and the drop of tong. Therefore,two palate shelves failed to fuse with each other.2. The effect of atRA on TGF-β/Smad2/3signaling pathways in CP:The expression of Smad2/3-positive cells increased gradually in the embryonic palatal mesenchymal cells(EPMs) from GD1318to GD148, while few positive cells were observed after GD158, and the level of the same developmental time was lower than that of the experimental group. After palate shelves contact,the expression of smad2/3-positive cells decreased in the medial edge epithelial(MEE). The same expression level of smad2/3occurred both vegetable oil control group and blank control group.ConclusionsThe changes of Smad2/3expression induced by excess atRA in the embryonic palatal medial edge epithelium and embryonic palatal mesenchymal cells may be related to the cleft palate through epithelial-mesenchymal transformation (EMT), thus atRA result in the formation of cleft palate.