| ObjetctiveThis article through the carrier beads implanted citral into the normal developmental nasal pit of the chick embryo, to inhibit the synthesis of retinoic acid and research the role of palatal retinoic acid during the process of the palate development. By detecting the expression change of FGF8surround the nasal pit after inhibition of retinoic acid, clarify the importance of FGF8during palate development, to reveal the relationship between exogenous factors and endogenous signaling system interaction during palate formation. It provides a new thought and scientific basis to explore the influence factor during palate construction.Methods Exogenous citral inhibition of retinoic acid synthesis in chick embryo effects on the palatal tissue morphology:160eggs were randomly divided into DMSO negative control group, normal control group and six experimental groups. According to the citral concentration from low to high the experimental groups were divided as0.00lg/ml,0.01g/ml,0.05g/ml,0.1g/ml,0.5g/ml,0.88g/ml. There were20eggs in each group. At stage20, the experimental group was implanted citral beads of different concentration to the nasal pit, negative control group was implanted DMSO beads, the normal control group was without any operation. Then the chick embryos were continued incubation to stage38.Throught hard and soft facial skeletal transparent staining, observe the effects on the palatal development, and find out the best citral concentration to induce the palatal defects. And the dose was used in the following experiments. Prepare another40eggs, which were randomly divided into two groups:the experimental group was implanted beads with the best citral concentration in stage20, the control group simultaneously was placed the DMSO beads, respectively. The chick embryos were continuing hatching to stage24,25,26,27,28,29,30,31. Each group was taken2embryos to observe the development and fusion of the facial process at each stage. Then the specimen were fixed, done the serial sections and HE stain for morphological observation.Exogenous citral inhibition of retinoic acid synthesis in chick embryo effects on the expression fibroblast growth factor8in palatal tissue:90eggs were randomly divided into three groups:experimental group, negative control group and normal control group. There were30eggs in each group. At stage20, the experimental group was implanted the citral beads with the upper concentration into the nasal pit, negative control group was implanted DMSO beads, the normal control group was without any operation, respectively. The chick embryos were continuing hatching to stage24,25,26,27,28. First of all,5embryos at stage24were randomly selected from the normal control group to determine the FGF8expression site in facial area by whole-mount immunohistochemistry. Then another5embryos were randomly selected in each group in different stage to be done the immunohistochemistry after serial sections. The FGF8expression changes around the nasal pit tissue were detected. The average value of optical density was statistical analysised.Exogenous citral inhibition of retinoic acid synthesis in chick embryo effects on the cell apoptosis in palatal tissue:30eggs were randomly divided into two groups:the experimental group and control group. There were15eggs in each group. At stage20, the experimental group were implanted the citral beads with the upper concentration into the nasal pit, while the control group were implanted DMSO beads to the nasal pit. The chick embryos were continuing hatching for3,6,16hours.5embryos were taken at each group and each time point to observe the local cell apoptosis through Nile blue staining.ResultsCraniofacial skeletal staining showed:The craniofacial growth hadn’t been influenced by the DMSO beads and0.00lg/ml,0.01g/ml and0.05g/ml citral. The maxillofacial bone defects were caused by0.1g/ml citral,but the palate was not affected. The nasal bone and maxillary bone absolute defects and the maxilla, zygomatic bone partial defects, and the palate defects at the treated side were all caused by0.5g/ml citral, the untreated side was intact. More severely defects were caused by0.88g/ml at treated side. All the bone almost entirely absent, the volume was significantly reduced, and the palate was severely impaired. The nasal bone was also defected at the untreated side, while the other bones showed no significant abnormality. The optimum concentration of Citral was0.5g/ml can stably lead to palatal defect. Effects on local by citral were rapidly. The defects of Stage24chick embryos at the treated side can be visible by HE stain. The treated side at stage26,27,28,29,30,31appeared growth retardation, and the volume of the lateral nasal processes was significantly reduced.The immunohistochemical results of stage24normal chick embryo show: FGF8expressed in the eye, brain and facial process. The positive cell was brown, deep dyeing. And the ectoderm of the eyes, brain and nasal pit had high expression, colored deeper.0.5g/ml citral beads were implanted in the chick nasal pit, the expression of FGF8was decreased rapidly and retained at low level during stage24,25,26,27,28.It was significantly lower than that of DMSO control group and normal control group.P<0.01, there was significant difference between citral group and other groups. While the expression curves of FGF8in DMSO group and normal control group were similar. They began to gradually increased from stage24, continued to show a high expression at stage26and27when was peak of development and fusion of the facial process. The expression of FGF8was decreased near stage28when the process fusion almost completed.P>0.05, there was no significant difference between the two groups.Nile blue staining showed that DMSO group had normal local cell apoptosis at3,6,16hours. Citral group showed no abnormal apoptosis in3hours, began appearing obvious apoptosis near the nasal pit in the6hours, the cell apoptosis was more serious in16hours. The cell apoptosis around the nasal pit increased as the time went on.ConclusionsThis study shows that:the local apoptosis and palatal defects in chick embryo were caused by citral application for inhibition synthesis of retinoic acid. Retinoic acid deceased may cause local growth restriction and the expression of FGF8declined in process of palatal development. The expression level of FGF8decreased may also increas the local apoptosis. The interaction of endogenous retinoic acid and FGF8can regulate cell growth and differentiation in the palate tissue, and played an important role in palate development. |
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