用体外器官培养方法研究EDCs对新生小鼠睾丸的影响

目的 通过已建立的小鼠睾丸体外培养系统，研究四种内分泌干扰物（Endocrine Disrupting Chemicals，EDCs）对男性内分泌系统的影响。 方法 将新生小鼠的睾丸组织在体外环境中培养24h，而后在培养基中分别加入浓度为0.1μM, 1μM, 10μM and 100μM的四种（DEHP、MEHP、NP、p, p’-DDE）内分泌干扰物并培养72h，同时设置对照组；培养结束后进行组织学观察，测定冻存培养基中睾酮和抑制素βB (INH-βB)的分泌水平，同时测定细胞色素P450侧链裂解酶(P450Scc)、3β-羟基类固醇脱氢酶(3β-HSD)、细胞色素P45017α-羟化酶(P450C17)和波形蛋白（vimentin）的基因表达情况。 结果 所有剂量组中睾酮的分泌水平均发生改变；P450Scc、3β-HSD、P450C17和INH-βB蛋白质的表达及mRNA水平均受到四种内分泌干扰物的影响（P<0.05）；DEHP和MEHP降低了波形蛋白的mRNA水平（P<0.05），而NP和p, p’-DDE对波形蛋白没有显著影响（P>0.05）。 结论 本研究建立的体外培养新生小鼠睾丸模型中，所选的四种已知EDCs改变了两种睾丸激素水平，三种类固醇合成酶以及与支持细胞功能相关的波形蛋白的表达。

Investigation of the effects of EDCs on neonatal mouse testis using vitro organ culture

Abstract: Objective To explore the effects of EDCs on the male endocrine through an established in vitro testis culture system. Methods Testicular tissues obtained from neonatal mice were cultured for 24 h in vitro and then treated with four EDCs， including di － ( 2 － ethylhexyl) phthalate ( DEHP) ，mono － 2 － ethylhexyl phthalate ( MEHP) ，nonyl phenol ( NP) and 1，1－ dichloro － 2，2 － bis ( p － chlorophenyl) － ethylene ( p， p’－ DDE) ，at concentrations of 0. 1 μM，1 μM，10 μM and 100μM for 72 h， and the control groups were set up. Histological evaluation was performed after the culture. The effects of EDCs on cultured neonatal mouse testes were investigated by the level of testosterone and inhibin βB ( INH － βB) ，as well as the gene expression of cytochrome P450 side － chain cleavage enzyme ( P450Scc) ，3β － hydroxysteroid dehydrogenase ( 3β －HSD) ，cytochrome P450 17 α － hydroxylase ( P450C17 ) ，vimentin. Results Changes in testosterone secretion were observed in all tested EDC groups， and both the protein expression and mRNA level ofP450Scc，3β － HSD，P450C17，andINH － βB were significantly affected by the four EDCs( P ＜ 0. 05) . DEHP and MEHP down － regulated the vimentin mRNA levels( P ＜ 0. 05) ，whereas NP and p， p’－ DDE had no obvious effect on vimentin( P ＞ 0. 05) . Conclusion These results suggest that four EDCs result in alterations of two testicular hormones， three steroidogenic enzymes and vimentin linked with the function of Sertoli cells in the cultured neonatal mouse testes model， which is an in vitro organ culture system.