TCDD对小鼠腭突间充质细胞增殖和凋亡的影响

Effects of 2,3,7,8-tetrachlorodibenzo-p-doxin on proliferation and apoptosis of mouse embryonic palatal mesenchymal cells

目的:探讨2,3,7,8-四氯二苯并对二噁英(TCDD)对小鼠腭突间充质细胞增殖和凋亡的影响。方法:采用随机数字表法将SPF级C57BL/6N孕鼠分为TCDD组和对照组,在妊娠第10天(GD10)分别以64μg/kgTCDD和等量玉米油灌胃,取GD13、GD14、GD15胚胎头部,行组织形态学观察。收集GD13正常胎鼠腭组织,分离腭突间充质细胞,用0.0、0.1、1.0、10.0nmol/LTCDD处理72h后,采用MTT法检测细胞增殖并计算增殖抑制率,检测Brdu阳性率,采用荧光定量RT-PCR和Westernblot法检测细胞中PCNA、Bax、Caspase-3mRNA和蛋白表达水平。结果:组织形态学显示TCDD组胎鼠双侧腭突未接触和融合。随着TCDD作用浓度的增加,小鼠腭突间充质细胞增殖抑制率逐渐增加,Brdu阳性率逐渐降低,细胞中Bax、Caspase-3mRNA和蛋白表达逐渐增加,而PCNAmRNA和蛋白表达逐渐降低(P<0.05)。结论:TCDD可能通过抑制腭突间充质细胞的增殖并促进其凋亡,导致腭裂的发生。

Aim :To investigate the effects of 2, 3, 7, 8-tetrachlorodibenzo-p-doxin(TCDD) on proliferation and apoptosis of mouse embryonic palatal mesenchymal(EPM) cell. Methods : The SPF-class C567BL/6N pregnant mice were allocated into TCDD group and control group randomly. On gestation day 10(GD10), pregnant mice were gavaged by TCDD at 64 μg/kg(TCDD group) and equal amount of corn oil(control group). Coronal sections of GD13 to GD15 embryos were taken and stained with HE to observe morphological changes.GD13 normal fetal mouse palatal tissue was collected,and EPM cells were isolated and cultured with different doses of TCDD(0.0, 0.1, 1.0, 10.0 nmol/L) for 72 hours, the cell proliferation was detected by MTT assay, and the Brdu positive rate was detected. Real-time RT-PCR and Western blot were used to determine the expression levels of PCNA, Bax, Caspase-3 mRNA and protein. Results : Morphological results showed bilateral shelves did not contact and fusion in TCDD group.With the increase of TCDD dose, the cell proliferation inhibition rate increased gradually, and the Brdu positive rate gradually decreased( P <0.05);the expressions of Bax, Caspase-3 mRNA and protein gradually increased, while the expression of PCNA mRNA and protein gradually decreased( P < 0.05). Conclusion : TCDD may cause cleft palate by inhibiting the proliferation of EPM cells and promoting apoptosis .