O-GlcNAc通过抑制Wnt信号通路影响上唇发育的实验研究

Experimental study of O-GlcNAc on the influence of the upper lip development through inhibition of Wnt signaling pathway

目的探讨氧联乙酰葡萄糖胺(O-linked N-acetylglucosamine,O-Glc NAc)影响上唇发育时上皮层的消失过程中,Wnt信号通路在其中发挥的作用。方法通过在鸡胚唇部植入浸有O-GlcNAc促进剂氧联乙酰葡萄糖胺转移酶(O-GlcNAc transferase,OGT)、O-GlcNAc的抑制酶氧联乙酰葡萄糖胺水解酶(O-Glc NAcase,OGA)以及OGA的抑制剂链脲佐菌素(streptozoticin,SZT)的琼脂糖微球,构建O-Glc NAc高表达与低表达以及反向高表达实验组,并继续孵育24 h,茜素红—阿利新蓝骨组织染色观察胚胎骨、软骨发生的变化,TUNEL法检测细胞凋亡情况,免疫组化染色法检测Wnt信号系包括GSK-3β、β-catenin在内的一系列信号因子。结果 O-GlcNAc过表达组OGT和STZ中,上皮层的消失出现延迟,骨染色发现胚胎唇部出现骨缺损,TUNEL检测与正常组相比未发现明显细胞凋亡,但GSK-3β在细胞质内表达增加,β-catenin细胞核内表达减少;正常侧及O-GlcNAc低表达组OGA中上唇发育未受影响。结论 O-GlcNAc过表达时引起上皮层的消失发生延迟,这并非由细胞凋亡减少引起,而是可能通过抑制Wnt信号通路,导致GSK-3β、β-catenin在内的相关信号因子变化,这些变化将影响上唇发育并可能导致唇腭裂的发生。

Objective To explore the role of Wnt signaling pathway in the process of epithelial layer＆#39;s disappearance when Oxydiethanoldiethylene GlcNAc influence development of upper lip. Methods The over/lower-expression of O-Gl-cNAc and reverse-high expression experimental group was constructed by transplanting beads into the upper lip of chicken embryo which contain O-GlcNAc promoter OGT （ O-GlcNAc transferase） , O-GlcNAc inhibitor OGA （ O-GlcNAcase） or OGA inhibitor STZ（ streptozoticin） . and kept incubation for 24 h. Phenotypic analysis of skeletal and morphological chan-ges were performed by using Alizarin Red/Alcian Blue method. TUNEL assay was performed to analyze the cell apopto-sis. Immunohistochemistry method was performed to test the Wnt signaling pathway including GSK-3β, β-catenin. Re-sults In the group OGZ and STZ where O-GlcNAc was overexpressed, disappearance of the epithelial layers was de-layed, embryo lips appeared bone defect in bonestaining, compared with the normal group, obvious cell apoptosis has not been found in TUNEL detection. But expression of GSK-3 beta increased in the cytoplasm, expression of beta-catenin nu-cleus reduced, upper lip development of normal side and the low expression of O-GlcNAc in the OGA group were not af-fected. Conclusion Overexpression of O-GlcNAc may lead to a delay of epithelial disappearance which was not caused by the apoptosis but maybe caused by inhibiting Wnt signaling pathway. The process can change related signaling factors including GSK-3 beta, beta-catenin, which may influence the development of upper lip and in the end lead to cleft lip and palate.