邻苯二甲酸二丁酯与邻苯二甲酸单丁酯对睾丸间质细胞睾酮水平和胰岛素样因子3表达的影响

Effects of dibutyl phthalate and monobutyl phthalate on testosterone secretion and insulin-like factor 3 expression of Leydig tumor cells in mice

目的观察邻苯二甲酸二丁酯（dibutyl phthalate，DBP）、邻苯二甲酸单丁酯（monobutyl phthalate，MBP）对小鼠睾丸间质瘤细胞（MA-10）中胰岛素样因子3（insulin．1ikefactor3，INSL3）mRNA及蛋白的表达量和细胞睾酮分泌水平的影响。方法以小鼠睾丸间质瘤细胞（MA-10）为模型，经过DBP、MBP直接染毒后，采用酶联免疫吸附法（ELISA）测定细胞培养基上清中睾酮的含量，采用荧光定量PCR测定细胞中INSL3mRNA的表达，采用蛋白免疫印迹法（western blot）测定INSL3蛋白的表达水平。结果与对照组比较，MA-10细胞睾酮合成量在低染毒剂量（10^-9～10^-6mol／L）时均表现出刺激效应，睾酮含量增加；在高染毒剂量（10^-3mol／L）时表现抑制效应，随着染毒时间的延长，睾酮水平逐渐降低。荧光定量PCR以及Western blot测定结果显示，DBP在10^-6、10^4mol／L染毒时，INSL3mRNA及蛋白表达水平较对照组降低，差异有统计学意义（P〈0．05）；随着MBP染毒剂量的增加，INSL3mRNA及蛋白表达水平呈现下降趋势，但MBP在10^-7mol／L时，与对照组相比，对MA-10细胞中INSL3蛋白表达表现出刺激作用。结论DBP、MBP染毒对MA-10细胞睾酮分泌表现出高剂量抑制、低剂量刺激的双向效应；DBP、MBP染毒可抑制MA-10细胞中INSL3mRNA和蛋白的表达。

Objective To observe the effectsofdibutyl phthalate （DBP） and monobutyl phthalate （MBP） on the mRNA and protein expression of insulin-like factor 3 （INSL3） in the Leydig tumor cells （MA-10） of mice and the level of testosterone secreted from MA-IO cells. Methods The MA-10 ceils of mice, used as a cellular model, were exposed to DBP and MBP. The content of testosterone in the supernatant medium was measured by enzyme-linked immunosorbent assay; the mRNA and protein expression levels of INSL3 in MA-10 cells were measured by quantitative PCR and Western Blot. Results Compared with the control group, MA-10 cells showed increased synthesis of testosterone when exposed to low concentrations of DBP and MBP （10^-9-10^-6mol/L） and inhibited synthesis of testosterone when exposed to high concentrations of DBP and MBP （ 10^-3 mol/L）, and the typical two-way effects became more significant as the time went one and the concentrations increased （P〈0.05）. Compared with the control group, MA-10 ceils showed significantly lower mRNA and protein expression levels of INSL3 when exposed to 10n and 10^-4 mol/L DBP （ P〈0.05 ）; MA-10 cells showed increased protein expression of INSL3 when exposed to 10^-7 mol/L MBP, and the mRNA and protein expression levels of INSL3 decreased as the concentration of MBP increased. Conclusion DBP and MBP can inhibit the secretion of testosterone from MA-10 cells at high concentrations, but stimulate the secretion of testosterone at low concentrations. Both DBP and MBP have inhibitory effects on the mRNA and protein expression of INSL3 in MA-10 cells.