γ-氨基丁酸A型受体β3亚基在2,3,7,8-四氯二苯并二英诱导胎鼠腭裂模型中表达的研究

Expression of gamma-aminobutyric acid type A receptor beta3 subunit in murine cleft palate induced by 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin

目的利用2,3,7,8-四氯二苯并二英(2,3,7,8-tetrachlorodibenzo-p-dioxin,TCDD)诱导C57BL/6J胎鼠腭裂,检测γ-氨基丁酸A型受体β3亚基(gamma-aminobutyric acidt ype Areceptor beta 3 subunit,GABRB3)在TCDD诱导胎鼠腭裂模型中的表达变化情况,探讨腭裂的发生机制。方法将60只孕鼠按随机数字表法分为实验组和对照组,每组30只。妊娠第10.5天(gestation day 10.5,GD10.5)实验组以28μg/kg的TCDD单次灌胃,对照组以5.6ml/kg玉米油单次灌胃。分别在GD13.5、GD14.5、GD15.5、GD16.5、GD17.5处死孕鼠12只,每组处死6只。行胎鼠腭部形态学和组织学观察;应用实时荧光定量PCR和蛋白质印迹法检测胎鼠腭部组织中GABRB3mRNA及蛋白表达;免疫组织化学及免疫荧光染色检测GABRB3蛋白的表达部位。结果实验组共36只胎鼠,GD17.5腭裂的发生率为100%(36/36);对照组共47只胎鼠,无腭裂发生(0/47)。实验组胎鼠腭突上抬在GD15.5完成,较对照组腭突上抬延迟1d;GD14.5、GD15.5、GD16.5、GD17.5实验组中GABRB3基因mRNA(分别为0.561±0.073、0.728±0.104、0.782±0.137、0.686±0.145)和蛋白表达(分别为0.288±0.013、0.404±0.017、0.399±0.012、0.307±0.010)均显著低于对照组GABRB3基因mRNA(分别为0.818±0.088、0.865±0.086、1.021±0.054、1.163±0.179)和蛋白表达(分别为0.481±0.017、0.456±0.009、0.474±0.016、0.529±0.015)(P<0.05);免疫组化及免疫荧光结果显示,GABRB3主要表达于腭突间质细胞和腭突中嵴上皮缝。结论TCDD诱导胎鼠腭突上抬延迟并最终导致腭裂发生可能与GABRB3降低有关;GABRB3可能在腭部发育的上抬和融合期发挥重要作用。

Objective To investigate the expression of gamma-aminobutyric acid type A receptor beta3 subunit (GABRB3) on cleft palate in C57BL/6J mice induced by 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD). Methods Sixty C57BL/6J pregnant mice on gestation day (GD) 10.5 were divided into two groups: one group was administered through gastric tubes one dose of 28 μg/kg TCDD (experimental group) and the other group was administered through gastric tubes one dose of 5.6 ml/kg corn oil (control group). Embryos were removed by cesarean section from pregnant mice during the palatal formation stage (GD 13.5-17.5) and the palatal tissue studied in morphological and histological observation. The relative mRNA and protein expression of GABRB3 was measured by real-time quantitative PCR and Western blotting. Localization of GABRB3 protein was measured by immunohistochemistry or immunofluorescence. Results The incidence of cleft palate at GD17.5 was 100% in experimental group and there was no cleft palate occurred in the control group (0);elevation of palatine processes in experimental group was completed on GD15.5 which was clearly delayed by a day compared with that in control group. On GD14.5-GD17.5, the mRNA expression (0.561±0.073, 0.728±0.104, 0.782±0.137, 0.686±0.145) and protein expression (0.288±0.013, 0.404±0.017, 0.399±0.012, 0.307±0.010) in the experimental group were significantly lower than the control group mRNA expression (0.818±0.088, 0.865±0.086, 1.021±0.054, 1.163±0.179) and protein expression (0.481±0.017, 0.456±0.009, 0.474±0.016, 0.529±0.015)(P<0.05). Immunohistochemistry and immunofluorescence showed that GABRB3 was mainly expressed in the mesenchymal cells and medial edge epithelium. Conclusions TCDD delayed palatal shelf elevation and eventually led to cleft palate may be associated with a decrease in GABRB3. GABRB3 may play an important role in the elevation and fusion phases of the palate development.