The Mechanism Of The Cleft Palate Inducing By 2,3,7,8-Tetrachlorodibenzo-p-dioxin In C57BL/6N Mouse

Cleft palate is one of the common birth defects in human.A series of dysfunctions such as eating and swallowing caused by cleft palate could bring mental and economic burden to patients and their families even the society.As a complex congenital disease,cleft palate can be induced by genes and environmental factors.However,the specific pathogenesis of cleft palate is still unknown.2,3,7,8-Tetrachlorodibenzo-p-dioxin(TCDD)is one of the most toxic dioxin compounds.As an environmental endocrine disruptor,TCDD can accumulate in the animal and human body through the environment and food,and TCDD can pass through the placental barriers,causing birth defects such as cleft palate and hydronephrosis.However,the specific mechanism of TCDD-induced embryo palate is unclear.TCDD exerts toxic effects mainly by activating the aromatic hydrocarbon receptor(AhR)pathway,and TCDD can also affect vitamin A metabolism in the body.Octamer-bingding transcription factor 4,Oct4,as an embryo-specific transcription factor,plays an important role in inhibiting cell differentiation and maintaining stem cell multipotency.Oct4 participates in signal transduction of pluripotent cells by activating proliferation-related factors,and can induce somatic cell dedifferentiation,and activate histone demethylase to participate in epigenetic regulation.Oct4 is also involved in the differentiation induced by all-trans retinoic acid(RA).In addition,there is an interaction between Oct4 and AhR.Studies have shown that Oct4 is biologically related to cleft palate,and there are AhR and RAR binding elements at the 5 'end of Oct4.In the early stages of this study,we found that TCDD could inhibit the expression of Oct4 in fetal mice palatal tissue,suggesting that Oct4 might play a role in TCDD inducing cleft palate.In this study,by establishing the TCDD-induced fetal mice palate model and the primary culture model of fetal mice palatal mesenchymal cells,the related mechanisms of TCDD-induced palate and the important role of Oct4 in animal experiments and cell experiments were studied.Researching on cleft palate mechanism and disease prevention might provide theoretical and experimental basis.Objectives1.Animal models of cleft palate and fetal mice palatal mesenchymal cells were used,and the effects of TCDD on the proliferation and apoptosis of palatal mesenchymal cells in vitro and in vivo were observed.2.By detecting the expression of key molecules in the RA signaling pathway,the effects of TCDD on the RA signaling pathway were explored.3.By analyzing the expression of Oct4 in fetal mice palatal mesenchyme,its binding with molecular of RA signal,and the mechanism of Oct4 in TCDD-induced palate was explored.4.By detecting the methylation level of promoter region of Oct4 gene at GD14,the possible mechanism of TCDD-induced palate was explored.MethodsExperiments in vivo1.Pregnant mice were randomly divided into experimental group and control group.The pregnant mice in the experimental group were orally administered with a dose of 64?g/kg�bw of TCDD once at about 10 am GD10,and the pregnant mice in the control group were orally administered with an equal volume of corn oil.Forty pregnant mice in the experimental group and the control group were obtained.2.Paraffin sections were obtained from part of fetal mice' heads of the obtained pregnant mice at GD13,GD14,and GD15.And HE staining,immunohistochemistry,and BrdU and TUNEL analysis were performed to observe the development of palates at GD13,GD14,and GD15.As well as the dynamic process of cleft palate induced by TCDD,and the effect of proliferation and apoptosis on fetal mice palatal mesenchyme.3.Palatal shelves of fetal mouse were taken to detect the effects of TCDD on the expression of Oct4 and major molecules in RA signaling pathways by Western Blot.4.Bisulfite sequencing PCR were used to detect the effect of TCDD on the methylation level of gene promoter region of Oct4 in palatal mesenchyme.5.Co-immunoprecipitation was used to detect the role of TCDD on Oct4 binding to RA relative proteins in fetal mice palatal mesenchyme.Experiments in vitroAfter isolation and identification of mouse embryonic palatal mesenchyme cells obtained from GD13 fetal mice,primary culture of embryonic palatal mesenchyme cells was performed.CCK8 experiment was used to detect the effects of different concentrations of TCDD on cell vitality of fetal palatal mesenchymal cell.Immunofluorescence,The BrdU and TUNEL methods were used to observe the effects of TCDD on the proliferation and apoptosis of palatal mesenchyme cells.The qRT-PCR and Western Blot were used to detect the effects of TCDD on the expression of major molecules in RA signaling pathway.Si RNA and Western blot was used to detect the effect of TCDD on CRABPII protein expression after Oct4 gene silencing.Results1.The incidence of cleft palate inducing by TCDD(64?g/kg�bw)was 95.9%.HE staining results showed that the fetal mice in the control group palatal shelves could normally grow up to the horizontal direction in the GD13-GD15,and the bilateral shelves could be completely fused.In the experimental group,the palatal shelves of the fetuses were delayed to uplift at GD14.And the shelves in the horizontal could not contact and fuse to form a clear fissure.2.The results of immunohistochemical staining,BrdU,TUNEL and Western Blot showed that TCDD can inhibit the proliferation and increase the apoptosis of palatal mesenchyme,comparing with the control group.The expression of proliferation-related proteins PCNA was decreased significantly and the expression of apoptosis-related proteins was promoted significantly by TCDD compared to control group in fetal mice palatal mesenchyme(P<0.05).In cell experiments,TCDD can inhibit the proliferation and vitality of palatal mesenchyme cells,and promote the apoptosis of mesenchymal cells(P<0.05).3.The results of qRT-PCR showed that the expression of Oct4 and molecules related to the RA signal pathway were inhibited by TCDD.4.The results of Co-immunoprecipitation showed there was a binding between Oct4 protein and RARB(CRABPII)protein.And the binding between them could be increased by TCDD.5.Bisulfite sequencing PCR results showed that TCDD did not change the methylation level of Oct4 promoter region at GD14(P>0.05).6.The results of Oct4 siRNA-transfected mesenchymal cells showed that the inhibitory effect of TCDD on CRABPII disappeared after Oct4 was silenced.As the concentration of TCDD increased,the expression of CRABPII was up-regulated.Conclusions1.TCDD could inhibit the proliferation and promote the apoptosis of fetal palatal mesenchyme,which might be the key mechanism of TCDD-induced cleft palate.2.TCDD might induce cleft palate by interfering RA signal pathway in the fetal palatal mesenchyme.3.Oct4 might play a role in TCDD-induced cleft palate and influencing the RA signaling pathway.4.The hypermethylation of Oct4 promoter region might not be the mechanism of TCDD reducing the expression of Oct4.