二甲磺酸乙烷对成年大鼠睾丸间质不同时间点细胞类固醇合成酶的影响

Effects of ethane dimethane sulfonate on the steroidogenesis enzymes of leydig cells in adult rats at different time-points

目的研究二甲磺酸乙烷(EDS)注射对雄性大鼠睾丸间质细胞类固醇关键合成酶表达的影响,并进一步了解EDS对大鼠睾丸间质细胞类固醇关键合成酶可能的调节机制。方法将25只90 d雄性SD大鼠随机分为两组,对照组(0 d,n=5)一次性腹腔内注射生理盐水,EDS组(n=20)一次性腹腔内注射EDS 70 mg/kg体重。EDS组注射后7、21、35、90 d后取血清和睾丸组织,用酶联免疫吸附法(ELISA)测定血清及睾丸中的睾酮水平,采用实时荧光定量PCR(q RT-PCR)和蛋白免疫印迹方法(Western blot)分别检测睾酮合成相关基因类固醇急性调节蛋白(St AR)、胆固醇侧链裂解酶(P450scc)、3β-羟基类固醇脱氢酶1型(3β-HSD1)、P45017α-羟化酶1(P450c17a1)、17β-羟基类固醇脱氢酶3型(17β-HSD3)m RNA和蛋白表达水平。结果与对照组比较,EDS处理7 d后,睾丸内的睾丸间质细胞几乎完全被破坏,导致睾酮水平急剧下降(P<0.01),睾酮合成相关酶St AR、3βHSD、P450scc、P450c17、17β-HSD3的m RNA及蛋白表达水平均显著下降(P<0.01),而处理21、35 d后,上述相关酶m RNA及蛋白表达水平均有所回升(P<0.01或P<0.05),处理90 d后,上述相关酶基因及蛋白的表达水平已经基本上恢复到与对照组相近的水平,差异无统计学意义(P>0.05)。结论 EDS能够特异性地破坏雄性SD大鼠睾丸组织中的睾丸间质细胞,EDS的此种效应对研究睾丸间质细胞缺失下睾丸功能的变化进而研究精子发生的内分泌调控均有较大意义。

Objective To study the effects of injection of ethane dimethane sulfonate （EDS） on the expression of key steroidogenesis enzymes of leydig cells in male rats, and to further understand the possible regulatory mechanism of EDS for the key steroidogenesis enzymes of leydig cells. Methods Twenty-five 90 d-old-male SD rats were randomly divided into 2 groups： control group （0 d, n = 5） was administered with normal saline for one-time, EDS group （n = 20） was injected with EDS 70 mg/kg body intraperitoneally. The serum and testis of rats in EDS group were collected after treatment for 7, 21, 35, 90 d. The enzyme linked immunosorbent assay （ELISA） was performed to measure the levels of testosterone in serum and testis. Quantitative real-time PCR （qRT-PCR） and Western blot were used to measure the expression of mRNA and proteins of steroidogenic acute regulatory protein （STAR） of related genes of testosterone syn- thesis, P450 cholesterol side-chain cleavage enzyme （P450sce）, 3β-hydroxy steroid clehydrogenase-1 （313-HSD1）, P45017α-hydroxylase-I （P450e17al）, 17β-hydroxy steroid dehydrogenase-3 （1715-HSD3） respectively. Results Compared with control group, after treatment for 7 d, the whole leydig cells were almost completely destroyed by EDS,which led to a sharp decline of the concentration of testosterone （P 〈 0.01）, the expression of mRNA and proteins of related enzymes of testosterone synthesis STAR, 313HSD, P450scc, P450c17, 17β-HSD3 was significantly decreased （P 〈 0.01）, while after treatment for 21, 35 d, the expression of mRNA and proteins of related enzymes mentioned above was raised more or less （P 〈 0.01 or P 〈 0.05）, after treatment for 90 d, the expression of mRNA and proteins of related enzymes mentioned above was almost restored to the levels of control group, there were no statistically significant differences （P 〉 0.05）. Conclusion EDS can destroy the leydig cells in the testis of male SD rats specifically. This effect of EDS has great significance to study the changes of testicular function under the lack of leydig cells and to further study the en- docrine regulation of spermatogenesis