地塞米松对腭胚突上皮细胞PAR复合体和细胞极性的影响

目的 探讨地塞米松（DEX）是否可以影响腭中嵴上皮细胞（MES）PAR极性复合体基因的表达,并进一步扰乱其细胞极性而影响腭融合。方法 将孕鼠随机分为对照组和DEX组,DEX组按6 mg·kg^-1腹腔注射地塞米松磷酸钠注射液,对照组注射0.9%氯化钠0.1 mL。在E13.5、E14.0、E14.5、E15.5、E17.5断颈处死孕鼠获取腭胚突,观察腭裂的发生情况,并通过苏木精-伊红染色、扫描电子显微镜观察腭上皮的形态改变,通过免疫荧光染色、蛋白质印迹及实时荧光定量聚合酶链式反应检测PAR3、PAR6、aPKC基因和蛋白的表达。结果 DEX组腭裂发生率为46.15%,对照组腭裂发生率为3.92%,DEX组的腭裂发生率高于对照组（χ²=24.335,P=0.00）。与对照组相比,DEX组腭胚突发育延迟且短小,腭中嵴上皮为非极性排列,只由单层的上皮细胞组成,腭胚突表面平坦,球状结构减少;PAR3和PAR6蛋白仅在腭上皮中表达,aPKC则表达于腭上皮和腭间充质中;PAR3、PAR6及aPKC基因的表达均减少。DEX在蛋白和基因水平下调PAR3、PAR6、aPKC的表达。结论 DEX可以导致腭胚突的生长发育延迟,并造成PAR极性复合体在蛋白和基因水平的表达下降,从而使MES极性丧失导致腭裂。

Influence of dexamethasone on the cell polarity and PAR complex of the embryonic epithelial cells in the palate

Objective This study aims to investigate whether dexamethasone (DEX) can down-regulate the PAR complex and disrupt the cell polarity in the palatal epithelium during palatal fusion. Methods Pregnant rats were randomly divided into control and DEX groups, which were injected intraperitoneally with 0.9% sodium chloride (0.1 mL) and DEX (6 mg·kg^-1), respectively, every day from E10 to E12. The palatal epithelial morphology was observed using hematoxylin and eosin staining and scanning electron microscopy. Immunofluorescence staining, Western Blot analysis, and real-time polymerase chain reaction were performed to detect the expression of PAR3, PAR6, and aPKC. Results The incidence of cleft palate in DEX group (46.15%) was significantly higher than that in control group (3.92%), and the difference was statistically significant (χ²=24.335, P=0.00). DEX can also retard the growth of the palatal shelves and the short palatal shelves. The morphology and arrangement of MEE cells changed from polarized bilayer cells to nonpolarized monolayer ones. Additionally, the spherical structure decreased, which caused the cleft palate. PAR3 and PAR6 were only detected in the palatal epithelium, and aPKC was expressed in the palatal epithelium and mesenchyme. DEX can reduce the expression levels of PAR3, PAR6, and aPKC in the protein and gene levels. Conclusion DEX can down-regulate the complex gene expression in the MEE cells, thereby destroying the cell polarity and causing cleft palate.