Study On Exosomes In The Procedure Of Cleft Palate Induced By TCDD

PART Ⅰ COMPARISON OF THE BIOLOGICAL BEHAVIORS OF PALATAL MESENCHYMAL AND EPITHELIAL CELLS INDUCED BY 2,3,7,8-TETRACHLORODIBENZO-P-DIOXIN IN VITROObjective:This study employed primary epithelial and mesenchymal cells as models in vitro to explore proliferation,migration,apoptosis and epithelial-to-mesenchymal transition with two different doses of TCDD(10nmol/L,100 nmol/L),contrasted with a control group without TCDD.Methods: Mesenchymal and epithelia cells derived from the palatal tissues of C57BL/6J fetal mice at 14.5 days of pregnancy were both divided into control group and experimental group.The experimental group was partially separated to two subparts with two different concentrations of TCDD intervention respectively(10 nmol/L and 100 nmol/L),comparing with the control group incubated with normal medium.The cell proliferative viability was determined by CCK8 assay.Transwell assay was used to detect the migration of cells about 24 h.Apoptosis was identified with an Annexin V/FITC/PI apoptosis kit.The EMT process of epithelial cells marked with Zo-1 and vimentin was detected by Immunofluorescence double staining and laser scanning confocal microscopy at 24 h and 72 h.Results : The primary epithelial cells were homogeneous in their cobblestone epithelial morphology,and the mesenchymal cells were fusiform or star shaped,with typical mesenchymal morphology.The marker Zo-1 was expressed by epithelial cells,while the marker vimentin was expressed by mesenchymal cells.With 10nmol/L TCDD,epithelial cell proliferation was significantly inhibited,whereas mesenchymal cell proliferation was obviously increased.With 100nmol/L TCDD,the proliferation of both epithelial cells and mesenchymal cells was significantly inhibited.The motility of epithelial cells treated with either 10nmol/L or100nmol/L TCDD was equal to that of the cells in the control group.The motility of mesenchymal cells in the TCDD group was increased after treatment with a low dose of TCDD while the motility of mesenchymal cells was decreased after treatment with a high dose of TCDD.The apoptosis rate in the 10nmol/L and 100nmol/L TCDD groups was the same as that in the control group for epithelial cells,while the apoptosis of mesenchymal cells in the TCDD group was stimulated by the higher dose of TCDD.Treatment with TCDD was accompanied by a decrease in the mesenchymal marker vimentin,indicating the inhibition of epithelial cell EMT.The degree of EMT inhibition increased as the dose of TCDD increased.Conclusion:we found the EMT process of primary palatal epithelial cells occurred automatically in vitro without helping bilateral palatal contact.With the low dose of TCDD,transformation of epithelial cells to mesenchymal cells was inhibited,and mesenchymal cell proliferation and migration were promoted.At high dose,mesenchymal cells decreased,preventing palate development,uprising and contact,while the EMT of epithelial cells decreased.Regardless of dose of TCDD,no impact on migration and apoptosis of epithelial cells was noted,but there was increased apoptosis of mesenchymal cell in a dose-dependent。PART Ⅱ COMPARISON OF THE BIOLOGICAL BEHAVIORS OF PALATAL MESENCHYMAL AND EPITHELIAL CELLS INDUCED BY EXOSOMES IN VITROObjective : To compare the impacts of exosomes derived from mesenchymal cells induced separately with 0nmol/L,10nmol/L TCDD bout proliferation,apoptosis,migration of palatal mesenchymal cells,and the EMT process of MEE.Methods: The third to seventh passages of mesenchymal cells taken from the palatal process of C57BL/6J fetal mice at 14.5 days were used as parental maternal cells for extracting exosomes.The control group used a medium with 0nmol/L TCDD,while the experimental group contained 10nmol/L TCDD.The cell supernatant was collected every 48 hours and the cells were passaged.The cell supernatant was used to collect exosomes according to the method of the QIAGEN kit.The vesicles were identified by transmission electron microscopy,West Blot,and particle size analysis.The exosomes were fluorescently labeled with PKH26 and incubated with mesenchymal and epithelial cells at a concentration of 100 μg/ml for 24 hours.The phagocytosis and uptake of exosomes were observed by a laser confocal microscope.Mesenchymal cells and epithelial cells were divided into two groups.The exosomes of the control group produced by normal mesenchymal cells(Exo-Control)and the exosomes of the experiment group produced by mesenchymal cells mediated with TCDD(Exo-TCDD)were treated at a concentration of 100 μg/ml.We used CCK8 and Edu to measure proliferation,scratch experiment and transwell to measure migration,cell flow cytometry and West Blot to measure apoptosis,and immunofluorescence laser confocal to measure the degree of EMT.The two roups of exosomes were sequenced to screen for differential miRNA.Result: The vesicles collected from the supernatant of mesenchymal cells were exosomes.The exosomes secreted by mesenchymal cells could be taken up by their own cells and epithelial cells.TCDD lead to the differential expression of miRNA of exosomes through interfering with mesenchymal cells.The Exo-TCDD inhibited the proliferation and migration of mesenchymal cells and promoted apoptosis compared with the Exo-Control.The Exo-TCDD curbed the EMT process of epithelial cells compared with the Exo-Control.Conclusion: The exosomes produced by mesenchymal cells can be taken up both by mesenchymal and epithelial cells.The impacts about mesenchymal and epithelial cells from Exo-TCDD are consistent with influences proposed by the TCDD directly.TCDD may induce cleft palate through exosomes.