转化生长因子β信号及细胞凋亡在叶酸预防腭裂中的作用

Folic acid rescue of ATRA-induced cleft palate by restoring the TGF-β signal and inhibiting apoptosis

目的研究叶酸(FA)通过抑制转化生长因子β(TGF-β)信号及细胞凋亡,预防全反式维甲酸(ATRA)诱导小鼠腭裂的机制。方法采用腭板器官体外培养法进行腭板的培养。实验分为FA组、ATRA组、ATRA+FA组共三组。分别培养24、48、72h后,苏木精-伊红染色观察各组腭板在不同时间点的融合情况,原位末端转移酶标记技术(TUNEL)检测腭板细胞的凋亡情况,免疫组化检测各组腭板中半胱天冬氨酸蛋白酶9(caspase-9)、TGF-β3和TGF-β受体RⅡ(TGF-βRⅡ)在各组腭板的表达。结果培养24、48、72h后,FA组腭板的融合数分别为3、8、8,ATRA+FA组腭板的融合数分别为3、6、7,而ATRA组几乎未见腭板融合,差异有统计学意义(P<0.05)。与FA组、ATRA+FA组相比,ATRA组细胞凋亡率和caspase-9表达上升(P<0.05),ATRA能明显抑制TGF-βRⅡ的表达(P<0.05),但对于TGF-β3的作用却较小。结论 FA能够有效预防ATRA诱导的腭裂。细胞凋亡及TGF-β信号与FA预防ATRA诱导腭裂相关。

Objective To study the mechanism of folic acid rescue of ATRA-induced Cleft Palate by restoring the TGF-β signal and inhibiting apoptosis. Methods Use palatal shelf organ culture. The experiment was divided into 3 groups, such as the FA group, ATRA group, ATRA＋FA group. After 24, 48, 72 hours, the palate shelves were stained with HE, and the numbers of palatal shelf fusion were observed in each experimental group. TUNEL assay was used to detect apoptosis. Immunohistochemistry staining was used to detect the expression of caspase-9, TGF-β3 and TGFβR Ⅱ in the palatal shelf in each experimental group. Results After cultured for 24, 48, 72 hours, the numbers of palatal shelf fusion were 3, 8, 8 in the FA group, and 3, 6, 7 in the ATRA＋FA group, compared with nearly no fusion in ATRA group, there was a significant difference （P〈0.05）. A higher apoptosis rate and caspase-9 in MEPM cells were detected in the ATRA group than in the FA or the ATRA＋FA group. Compared with the FA and the ATRA＋FA group, ATRA had little effect on TGF-β3 in MEPM cells but significantly inhibited TGF-β receptor Ⅱ. Conclusion Folic acid can rescue the cultured palates to continue developing and fusing that were inhibited and resulted in cleft palate by ATRA. Apoptosis and TGF-β signaling in MEPM cells were involved in folic acid rescued ATRA-induced cleft palate.