尿道下裂子鼠生殖结节中的间充质-上皮转化作用  

Mesenchymal-epithelial transition of fibroblast cells in genital tubercles of hypospadiac male rats with utero exposure to di-n-butyl phthalate

作　　者：李晋昊 李祥[2] 张亚[2] 周云[1] 

机构地区：[1]苏州大学附属儿童医院泌尿外科,215025 [2]苏州大学附属儿童医院儿科研究所,215025

出　　处：《中华实验外科杂志》2017年第7期1173-1176,共4页Chinese Journal of Experimental Surgery

基　　金：江苏省临床医学科技专项一新型临床诊断技术攻关项目（BL2012051）

摘　　要：目的观察尿道下裂子鼠生殖结节中成纤维细胞的间充质-上皮转化现象，探讨邻苯二甲酸二丁酯（DBP）导致尿道下裂的机制。方法取孕SD大鼠，随机分为DBP组和对照组。于怀孕第13～19天，DBP组按照800 mg/kg体重DBP溶入玉米油（DBP和玉米油总剂量为3 ml/kg体重）后灌胃，对照组按照3 ml/kg体重玉米油灌胃。怀孕19 d取出子鼠，鉴别出DBP组中发生尿道下裂的雄性子鼠及对照组中的雄性子鼠并分为尿道下裂组和正常对照组，提取两组子鼠的生殖结节。用组织块法原代培养子鼠生殖结节中的成纤维细胞，倒置显微镜观察细胞形态变化，划痕实验检测细胞迁移能力变化，实时定量反转录聚合酶链反应（RT-qPCR）检测E-钙黏蛋白（E-cadherin）、波形蛋白（Vimentin）的mRNA表达，Western blot检测E-cadherin、Vimentin的蛋白表达。结果对照组的尿道开口于生殖结节顶端，而尿道下裂组的尿道开口于生殖结节腹侧，肛门生殖结节距离[（1.43±0.04） mm]明显小于对照组[（2.27±0.09） mm，P=0.023]。子鼠尿道下裂发生率为40.5%。与对照组比较，尿道下裂组生殖结节成纤维细胞形态发生了变化，细胞呈多边型，丝状伪足变短，细胞排列较紧密，平均迁移距离缩短[12 h：（0.17±0.04） mm比（0.14±0.03） mm、24 h：（0.26±0.05） mm比（0.20±0.04） mm，P=0.041]，E-cadherin的mRNA和蛋白相对表达量增加、Vimentin的mRNA和蛋白相对表达量降低（E-cadherin、Vimentin mRNA相对表达量：1.00±0.00比1.43±0.31（P=0.034）、1.00±0.00比0.51±0.12（P=0.031）；E-cadherin、Vimentin蛋白相对表达量：0.15±0.06比1.59±0.84（P=0.005）、1.88±0.45比0.30±0.13（P=0.003）。结论DBP在诱导子鼠发生尿道下裂的过程当中，促进了子鼠生殖结节中成纤维细胞的间充质-上皮转化作用，这可能与导致尿道下裂的发生有关。Objective To explore the mechanism in di-n-butyl phthalate （DBP）-induced hypospadias, we research mesenchymal-epithelial transition （MET） of fibroblast cells in genital tubercles of hypospadiac male rats with utero exposure to di-n-butyl phthalate.Methods Pregnant rats were randomly divided into two groups. Dams were treated by gavage daily from gestation day （GD） 13 to GD19 with corn oil vehicle （3 ml/kg） or DBP in corn oil at 800 mg/kg （total DBP and corn oil at 3 ml/kg）. On GD19, fetuses were immediately removed from the uterus, and sexed by internal examination of the reproductive organs. The hypospadiac rats and the normal rats were randomly divided into two groups （hypospadiac group and control group）. The AGD （anogenital distance） was measured. The incidence of hypospadias was calculated. Primary culture of fibroblast cells in genital tubercles were cultured under full aseptic precautions. The fibroblast cells were observed the cell shape under an inverted microscope and detected migration in scratch-wound experiment. Western blotting, real-time quantitative reverse transcriptase-polymerase chain reaction （RT-qPCR） were used to detect the expression of E-cadherin、Vimentin protein and mRNA in fibroblast cells with primary culture by tissue-pieces.Results The urethral orifice of hypospadias locates on the ventral surface of the GT （genital tubercle） and the urethral orifice of normal rat was near the tip of the GT. AGD in the hypospadiac group [（1.43±0.04） mm] was significantly lower when compared with the control group [（2.27±0.09） mm, P=0.023], and the incidence of hypospadias was 40.5%. DBP up-regulated mRNA and protein expression of E-cadherin, suppressed mRNA and protein expression of Vimentin, and caused a complete MET-phenotype, which manifested as the degeneration of large filopodia-like processes, spindle-cell shape and cell migration. Compared with the fibroblast cells of control group, the hypospadiac group showed significantly different cell migrati

关 键 词：子鼠 尿道下裂 邻苯二甲酸二丁酯 成纤维细胞 间充质-上皮转化 

分 类 号：R726.9[医药卫生—儿科]