成纤维细胞在邻苯二甲酸二丁酯诱导雄性胎鼠发生尿道下裂中的作用

Role of fibroblast cells in di-n-butyl phthalate-induced hypospadias in male SD rats

目的探讨邻苯二甲酸二丁酯（DBP）致大鼠发生尿道下裂的作用和可能机制。方法孕SD大鼠14只随机均分为DBP组和对照组。于妊娠第13~19天,DBP组采用DBP 800mg/kg溶入玉米油灌胃,对照组给予玉米油灌胃。妊娠第19天取出胎鼠,鉴别胎鼠性别,并分为尿道下裂组和对照组。提取两组胎鼠的生殖结节;原代培养生殖结节中的成纤维细胞,Western blot法检测同型雄激素受体A（AR-A）和AR-B以及Ⅰ型胶原蛋白的表达,qPCR检测AR mRNA表达,细胞增殖实验检测细胞增殖能力变化。结果 DBP组雄性仔鼠35只,有尿道下裂鼠12只（34.3%）;对照组雄性仔鼠41只,无尿道下裂鼠。对照组的尿道开口于生殖结节顶端,尿道下裂组胎鼠的尿道开口于生殖结节腹侧。与对照组比较,尿道下裂组胎鼠的肛门-生殖结节距离短[（1.57±0.09）mm vs.（2.45±0.16）mm],AR-B相对表达量低（细胞质：0.16±0.07vs.3.46±0.98;细胞核：0.13±0.07vs.2.17±0.58）,增殖能力下降,Ⅰ型胶原蛋白相对表达量低（0.22±0.09vs.1.27±0.78）（P〈0.05）。结论 DBP可降低胎鼠生殖结节成纤维细胞中AR-B和Ⅰ型胶原蛋白相对表达量,降低成纤维细胞的增殖能力,这可能会影响雄性胎鼠生殖器的发育导致尿道下裂的发生。

Objective To explore the role and underlying pathogenesis in di-n-butyl phthalate（DBP）-induced hypospadias in SD rats.Methods Forteen pregnant rats were randomly divided into two groups.The rats in group A were treated daily by gastric gavage with DBP 800mg/kg in corn oil.On the 19 th day of gestation,the fetuses were obtained and gender was identified,of which male fetuses were assigned into groups of C（with hypospadias）and D（without hypospadias）.The genital tubercles were taken out for primary culture of fibroblast cells.The expressions of homotype androgen receptor-A（AR-A）,AR-B and collagen type Ⅰ were detected by Western blot and qPCR.The proliferation of fibroblast cells was determined using MTT assay.Results Thrty-five male fetuses were identified from group A,of which 12（34.3%）fetuses were with hypospadias.Forty-one male fetuses were identified from group B,of which none had hypospadias.Urethral orifice of the rats in group D located at the top of the genital tubercle,which in group C located at the ventral side of the genital tubercle.Compared to group D,the rats in group C had a shorter distance from the anus to the genital tubercle[（1.57±0.09）mm vs.（2.45±0.16）mm],lower AR-B protein expression（0.16±0.07 vs.3.46±0.98 for the cell cytoplasm and 0.13±0.07 vs.2.17±0.58 for the cell nucleus）,less ability of proliferation,and lower expression of collagen typeⅠ protein（0.22±0.09 vs.1.27±0.78）（P〈0.05）.Conclusion DBP can decrease the expressions of AR-B and collagen typeⅠin fibroblast cells and inhibit the proliferation of fibroblast cells,which affects the development of genital tubercle and induces hypospadias formation in SD rats.