二甲磺酸乙烷对大鼠睾丸胚胎型Leydig细胞的作用

Effects of ethane dimethane sulfonate on fetal Leydig cells in neonatal rats

目的:评价细胞毒性药物二甲磺酸乙烷对新生大鼠睾丸胚胎型Leydig细胞的作用效应及安全使用剂量。方法:选取出生后3 d的雄性SD幼鼠共40只,分为对照组(二甲基亚砜溶剂组)、二甲磺酸乙烷75 mg/kg组、二甲磺酸乙烷100 mg/kg组和二甲磺酸乙烷125 mg/kg组,每组10只。予腹腔内注射不同实验剂量的二甲磺酸乙烷,注射后4 d分别处死幼鼠;测量动物体重及睾丸重量,放射免疫法检测血清中睾酮水平,睾丸组织冰冻切片行3β-羟基类固醇脱氢酶(3β-hydroxysteroid dehydrogenase,3β-HSD)染色,实时荧光定量PCR技术及Western blot技术检测睾丸组织中Leydig细胞相关特异基因及蛋白的表达水平。结果:药物注射后4 d,与对照组相比较,在血清睾酮水平方面[(0.542±0.117)μg/L,(0.124±0.021)μg/L,(0.113±0.015)μg/L,vs.(0.834±0.172)μg/L,P<0.05],75 mg/kg剂量组轻度下降,而100 mg/kg剂量组和125 mg/kg剂量组则明显降低;睾丸组织3β-HSD染色显示:75 mg/kg剂量组仍可见少量的3β-HSD染色呈阳性的胚胎型Leydig细胞(fetal leydig cell,FLC),100 mg/kg剂量组和125 mg/kg剂量组无3β-HSD染色呈阳性的FLC存在,但125 mg/kg剂量组睾丸曲细精管细胞排列严重紊乱;RT-PCR及Western blot检测显示:100 mg/kg剂量组睾丸组织Hsd3b1和Cyp17a1的mRNA表达水平及其相应蛋白表达水平明显降低(P<0.001)。结论:二甲磺酸乙烷能够特异性地破坏新生雄性SD大鼠睾丸组织中的胚胎型Leydig细胞,腹腔注射100 mg/kg二甲磺酸乙烷可建立起稳定、可靠的大鼠睾丸去胚胎型Leydig细胞模型。

Objective： To investigate the effects of ethane dimethane sulfonate （EDS） on fetal Leydig cells （FLCs） in neonatal rats. Methods： In the study, 40 male neonatal SD rats were divided into con- trol group and experimental groups. The rats in the experimental groups aged 3 days （PND3） were intra- peritoneally injected with one single EDS （75, 100 and 125 mg/kg）. The samples were collected on PND7. The body and testes was weighed, and the serum level of testosterone was detected. One testis was for histological analysis （3β-hydroxysteroid dehydrogenase stainining）, and the other was frozen in refrigerator for molecular determination （ RT-PCR, Western blot）. Results： Compared with the control group, on day 4 after EDS treatment, significant decrease of serum testosterone was observed in the 75, 100 and 125 mg/kg experimental groups [ （0.542 ±0. 117） μg/L, （0. 124 ±0.021） μg/L, （0. 113± 0.015） μg/L, vs. （0.834 ±0. 172） μg/L, P 〈0.05]. Meanwhile, fetal Leydig cells in dusters disap- peared with a lower expression level of Hsd3bl and Cypl7al after EDS treatment in the testes of neonatal male rats in EDS （100 mg/kg）-treatment group （P 〈0.001 ）. Conclusion： Ethane dimethane sulfonate can specifically deplete the fetal Leydig cells in testes of neonatal rats. Thus we could establish the FLCs＇ depletion model to know more about these testicular interstitial cells.