邻苯二甲酸二(2-乙基)己酯对原代培养小鼠胚胎睾丸Leydig细胞功能影响研究

Effect of Di(2-ethylhexyl)phthalate on Leydig cells from embryonic mouse testis

目的探讨邻苯二甲酸二(2-乙基)己酯[D i(2-ethylhexyl)phthalate,DEHP]对体外培养的小鼠胚胎睾丸间质细胞(Leyd ig细胞)3β-羟基类固醇脱氢酶(3-βHSD)活性及睾酮合成的影响。方法小鼠胚胎Leyd ig细胞原代培养。DE-HP染毒剂量分别为25、50、100、200 mg/L,通过检测睾酮合成关键酶3β-HSD活性、放免法测定培养液中睾酮浓度,研究DEHP对Leyd ig细胞功能影响。结果小鼠胚胎Leyd ig细胞分离培养后,纯度和存活率分别达(81.17±2.32)%、(80.88±2.80)%。DEHP(25 mg/L)处理12 h,或DEHP(50 mg/L)处理6 h后,实验组与对照组比较3β-HSD酶活性明显降低(P<0.05),随着时间延长和药物浓度增加差异更显著(P<0.01)。DEHP50、100 mg/L实验组,在12、24 h的睾酮水平明显低于对照组(P<0.05)或(P<0.01),存在时间-效应关系;DEHP 200 mg/L实验组在6、12、24 h与对照组比较均有非常显著差异(P<0.01),并可观察到Leyd ig细胞明显的形态学改变。结论DEHP能影响原代培养小鼠胚胎Leyd ig细胞3β-羟基类固醇脱氢酶活性,并抑制睾酮合成,存在时间-效应和剂量-效应关系。

Objective To explore the effect of Di（2-ethylhexyl）phthalate （DEHP） on testosterone secretion and activity of 3β-hydroxysteroid dehydrogenase （3β-HSD） in Leydig cells cultured from the embryonic mouse testis. Methods The Leydig cells were isolated from fetal mouse testis and were cultured in DMEM/ F12 medium. The identification and purity of Leydig cells were assessed by detecting 3β-HSD. After the Leydig cells were treated with 25, 50, 100,200 mg/L DEHP for 6, 12 or 24 h, the cell viability was measured by using trypan blue, and the 3β-HSD activity and the testosterone level in the medium of the cultured Leydig cells by radioimmunoassay were measured. Results The purity and viability of Leydig cells were （81.17 ± 2.32 ） % and （ 80. 88 ± 2. 80） %. Activities of the steroidogenic enzymes 3β-HSD decreased significantly when treated with 25 mg/L or 50 mg/L DEHP for 12 h or 6 h as compared to controls （P 〈0.05）, and decreased more significantly when DEHP concentration increased and treatment time prolonged （P 〈 0.01 ）. The testosterone level decreased significantly when treated by 50 mg/L and 100 mg,/L DEHP for 12 and 24 h as compared with the control group （P 〈0.05 or P 〈0. 01 ） in a time-dependent and dose-dependent effect. The 3β-HSD activity and the testosterone level of the Leydig cells were of significant difference when treated by 200 mg/L DEHP as compared with the controls, with obvious morph changes of Leydig cells. Conclusion DEHP affected the activity of 3β-HSD enzyme and Leydig cell proliferation. DEHP suppressed testosterone synthesis of the fetal mouse Leydig cells in primary culture in a dose-dependent and time-dependent effect.