Changes Of Palatal Cell Proliferation And Differentiation In Retinoic Acid Induced Cleft Palate Of Mouse At The Terminal Stage Of Embryonic Development

Background:All-trans retinoic acid (all-trans retinoic acid, atRA) is a vitaminderivative, has been shown to have significant teratogenic effects.in the early stage ofembryonic palate development, maternal intake excessive retinoic acid may lead to thedecreased proliferation of palatal mesenchymal cells and produce cleft palate. But at theterminal stage of secondary palate, particularly in palatal mesenchymal cells, the degreeof proliferation and differentiation has not been reported.Objective:To investigate the palatal epithelial and mesenchymal cell proliferationand differentiation level in retinoic acid induced cleft palate at Embryonic18.5days, byusing BrdU marked slow-cycling long term label-retaining cells (LRCs), and combinationof cell proliferation and differentiation markers, such as PCNA, Sox-2and P75.Methods:10weeks ICR pregnant mouse of SPF level were selected. Atembryonic10days,100mg/kg body weight retinoic acid was gavaged to pregnant miceto establish an cleft palate animal model. At the embryonic12days,80mg/kg bodyweight pregnant mice peritoneal injection of BrdU, to mark the low split slow cyclestatus cells in embryos18.5days. At Embryo18.5days, the cleft palate group andcontrol group mouse were perfused dehydration embedding, by using multipleimmunofluorescence to detect the distribution of BrdU and Sox-2, P75andPCNA-positive cells; Embryonic palatal epithelial and mesenchymal tissues wasdissociated and mRNA was isolated individually, the expression levels of Sox-2and75were detected by real-time quantitative PCR.Results:(1) At E18.5, in control group double staining of BrdU and PCNAshowed BrdU located in mesenchymal cells of the median palatine suture as well as theosteoblasts of ossification center, and PCNA located in basal layer of palatal epitheliumand scattered in the palatal mesenchymal cells, and with almost non-overlapping to BrdU-positive cells; bur in cleft palate group, a large number of BruU positive cellswidely distributed in the mesenchyme of the distal palate, but rare PCNA positive cells;the majority of osteoblasts in the ossification center was BruU and PCNA doublepositive.(2)Sox-2-positive cells located in the palate epithelium. However, there arepositive cells that occur in minute amounts in the palatal mesenchymal withoutoverlaping with BrdU. The expressed level of cleft palate group is higher than normalgroup with statistical significance.(3)P75mainly expressed in the palatal mesenchymal cells, and there isnon-overlapping between it with BrdU. The higher expressed level in cleft palate isrelative to the normal group, with statistical significance.Conclusion:At the terminal stage of embryonic development, in the cleft palatemouse induced by the retinoic acid the cell proliferation of palatal mesenchymal cellsdecreased. But the distribution of neural crest-derived cells with differentiationpotential and slow cycling cells provides a basis for the regeneration and repair ofcongenital cleft palate.