Objective: To explore the molecular mechanism of cleft palate in mice induced by 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD). Methods: The pregnant mice were randomly divided into TCDD-treated group (n=42) and control group (n=42). TCDD-treated group was given by gavage a single dose of TCDD (64 μg/kg) at 8: 00 AM on gestation day 10 (GD10) and the control group was given by gavage the isopyknic corn oil. At GD13-GD15, the fetal mice palate development was observed by HE staining. The mouse embryonic palatal mesenchymal cell proliferation was detected by 5-bromo-2-deoxyuridine (BrdU) immunofluorescence. The localization and expression of maternally expressed gene3 (MEG3) in mouse embryonic palatal mesenchymal cells was detected by situ hybridization and real-time PCR (RT-PCR). The key protein expressions of transforming growth factor-β (TGF-β)/Smad signaling pathway in mouse embryonic palatal mesenchyme were analyzed by Western blotting. The interaction of MEG3 and TGF-β receptor Ⅰ (TGF-βRⅠ) was examined by RNA binding protein immunoprecipitation (RIP). Results: At GD13 and GD14, compared with the control group, the ratio of BrdU-positive cells in the palatal mesenchyme of TCDD-treated fetuses decreased significantly (GD13, t=6.66, P=0.003; GD14, t=6.56, P=0.003). However, at GD15, the ratio of BrdU-positive cells was significantly increased (t=-5.98, P=0.004). MEG3 was mainly expressed in the nuclei of fetal mouse palatal mesenchymal cells, and the expression of MEG3 in TCDD group was significantly increased at GD13, GD14 and GD15(GD13, t=39.28, P=0.012; GD14, t=18.75, P=0.042; GD15, t=28.36, P=0.045). At GD14, TCDD decreased the levels of p-Smad2 and Smad4 in embryonic palate mesenchymal cells (p-Smad2, t=9.48, P=0.001;Smad4, t=63.10, P=0.001), whereas the expression of Smad7 was significantly increased at GD14 (t=30.77, P<0.001). The results of the RIP experiment showed that the amount of TGF-βRⅠ-bound MEG3 in mouse embryonic palatal mesenchymal cells in the TCDD group (23.940±1.301) was higher than that in the control group (8.537±1.523)(t=24.55, P<0.001). Conclusions: MEG3 is involved in the suppression of mouse embryonic palatal mesenchymal cell proliferation, functioning at least in part via interacting with the TGF-βRⅠ protein and thereby suppressing Smad signaling in the context of TCDD induced cleft palate.
目的： 探索2，3，7，8-四氯二苯并二(牙合)英（2，3，7，8-tetrachlorodibenzo-p-dioxin，TCDD）诱发小鼠腭裂的作用及机制。 方法： 将84只孕鼠根据体质量按随机数字表随机分为TCDD组和对照组（每组42只），在孕期第10天（gestation day 10，GD10）上午8时分别给予64 μg/kg TCDD和等量玉米油灌胃。HE染色观察GD13~GD15胎鼠腭发育的形态学变化，5-溴脱氧尿嘧啶核苷（5-bromo-2-deoxyuridine，BrdU）免疫荧光观察TCDD对GD13~GD15胎鼠腭突间充质细胞增殖的影响，原位杂交和实时定量PCR（real-time PCR，RT-PCR）检测母系印记基因3（maternally expressed gene3，MEG3）在胎鼠腭突间充质细胞中的定位和表达，蛋白质印迹法检测胎鼠腭突间充质细胞内Smad2、磷酸化Smad2（phospho-Smad2，p-Smad2）、Smad4、Smad7的蛋白表达，RNA 结合蛋白免疫沉淀实验（RNA binding protein immunoprecipitation，RIP）验证MEG3与转化生长因子β受体Ⅰ（transforming growth factor-β receptor Ⅰ，TGF-βRⅠ）的相互作用。 结果： 与对照组相比，TCDD组GD13（t=6.66，P=0.003）和GD14（t=6.56，P=0.003）胎鼠腭突间充质细胞中BrdU阳性细胞率显著减少，但在GD15时BrdU阳性细胞率显著增加（t=-5.98，P=0.004）。原位杂交显示MEG3主要表达于间充质细胞的细胞核。RT-PCR结果显示，TCDD组腭突间充质细胞内的MEG3相对表达量显著高于对照组（GD13：t=39.28，P=0.012；GD14：t=18.75，P=0.042；GD15：t=28.36，P=0.045）。在GD14，TCDD组胎鼠腭突间充质细胞内p-Smad2、Smad4蛋白表达均显著低于对照组（p-Smad2：t=9.48，P=0.001；Smad4：t=63.10，P=0.001），Smad7蛋白表达量显著高于对照组（t=30.77，P<0.001）。RIP实验结果显示，TCDD组GD14胎鼠腭突间充质细胞中TGF-βRⅠ结合MEG3的表达量（23.940±1.301）显著高于对照组（8.537±1.523）（t=24.55，P<0.001）。 结论： TCDD可能通过促进MEG3与TGF-βRⅠ的靶向结合影响TGF-β/Smad信号途径的激活，从而抑制腭突间充质细胞的增殖，由此导致腭裂的发生。.