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研究目的:通过四氯二苯并对二恶英(TCDD)和地塞米松(DEX)联合致畸构建更稳定的腭裂动物模型,初步研究维生素B12拮抗剂对腭突发育的影响,检测信号分子变化,为后续研究提供参考。创新要点:TCDD和DEX联合致畸可建立更稳定、更具研究价值的腭裂小鼠模型。从腭裂发生率、腭突发育形态大小、中嵴上皮细胞形态以及转化生长因子-β3(TGF-β3)、受体活化样激酶5(ALK5)分子表达等不同层次初步探讨了维生素B12的拮抗作用。研究方法:在小鼠腭发育关键时期,采用高剂量TCDD和DEX一次给药建立模型,并观察维生素B12的拮抗作用(见图3)。于胚胎期17.5天(GD 17.5)体视显微镜下检测各组腭裂发生率(见表1和图2),并于GD 13.5、GD 14.5、GD 15.5分别剪取胎鼠腭突提取RNA,采用实时荧光定量聚合酶链反应检测TGF-β3和ALK5基因表达(见图4、5)。重要结论:TCDD和DEX联合作用可诱导C57BL/6J胎鼠形成稳定腭裂。维生素B12对联合致畸诱导的TGF-β3及ALK5表达的改变及腭突中嵴上皮细胞形态的改变有一定程度的抑制作用,对腭裂发生率及腭突发育形态大小改变尚无明显抑制作用。
OBJECTIVE: To establish a more stable cleft palate animal model by combination of tetrachlorodibenzo-p-dioxins (TCDD) and dexamethasone (DEX), to study the effects of vitamin B12 antagonist on the development of palatal palate. The detection signal Molecular changes for the follow-up study provide a reference. Innovative points: TCDD and DEX teratogenic joint establishment of more stable and more research value of the cleft palate mouse model. The antagonism of vitamin B12 was initially explored at different levels, including the incidence of cleft palate, the size of the palatal epithelium, the morphology of the mid-crest epithelial cells, and the expression of transforming growth factor-β3 (TGF-β3) and receptor activated kinase 5 (ALK5) effect. Research Methods: In the critical period of mouse palatal development, high-dose TCDD and DEX were used to establish a model and observe the antagonism of vitamin B12 (see Figure 3). The incidence of cleft palate (Table 1 and Figure 2) was examined under a stereomicroscope at embryonic day 17.5 (GD 17.5), and the cleaved palate was extracted at GD 13.5, GD 14.5 and GD 15.5 respectively for RNA extraction. Real- Fluorescent quantitative polymerase chain reaction to detect TGF-β3 and ALK5 gene expression (see Figure 4,5). Important conclusion: TCDD and DEX can induce stable cleft palate in C57BL / 6J fetus. Vitamin B12 can inhibit the changes of the expression of TGF-β3 and ALK5 induced by joint teratogenicity and the morphological changes of the epithelial cells in the palatal midgut, but has no obvious inhibitory effect on the incidence of cleft palate and the size of the palatal development.