膜联蛋白5对大鼠睾丸间质细胞睾酮分泌及3β-羟基类固醇脱氢酶表达的影响

Effect of annexin 5 on testosterone secretion and expression of 3β-hydroxysteroid dehydrogenase in rat Leydig cells

目的先前研究发现促性腺激素释放激素(gonadotropin-releasing hormone,GnRH)刺激Leydig细胞后,膜联蛋白5(annexin 5)及睾酮水平同时增加,推测annexin 5和睾酮之间可能存在某种内在的联系,3β-羟基类固醇脱氢酶(3β-HSD)是间质细胞的标记酶。文中旨在研究annexin 5对大鼠睾丸间质细胞睾酮分泌及3β-HSD表达的影响。方法原代培养大鼠睾丸间质细胞,用不同浓度的annexin 5(0、10-10、10-9、10-8mol/L)处理间质细胞24h和用10-9mol/L的annexin 5处理细胞不同时间(6、12、24、36h),收集培养液,用化学发光法测定培养液中的睾酮含量;Western blotting方法分析睾丸间质细胞中3β-HSD的表达变化。结果不同浓度的annexin 5处理间质细胞24h后,与对照组比较,终浓度为10-10mol/L和10-9mol/L时,睾酮水平分别升高了18%[(14.38±0.30)nmol/Lvs(12.12±0.74)nmol/L](P<0.01)和26%[(15.25±0.47)nmol/Lvs(12.12±0.74)nmol/L](P<0.01);浓度为10-8mol/L时,睾酮水平虽也有升高趋势,但无显著性差异;而Western blotting结果显示,浓度为10-9mol/L时,3β-HSD表达量升高了63%(2.19±1.00vs1.34±0.19,P<0.05),浓度为10-10mol/L和10-8mol/L时,3β-HSD表达量无显著性差异;睾酮分泌与3β-HSD的表达呈显著正相关(r=0.430,P<0.05)。10-9mol/L的an-nexin5处理细胞不同时间,与对照组相比,6h和12h后睾酮的分泌虽有升高,但无统计学意义(P>0.05);24h后睾酮的分泌量显著升高了18%[(12.59±0.41)nmol/Lvs(10.70±0.09)nmol/L](P<0.01);而处理36h后,睾酮分泌量下降了21%[(14.44±1.65)nmol/Lvs(17.46±0.31)nmol/L](P<0.01)。结论在大鼠睾丸间质细胞中,annexin 5对睾酮分泌具有调节功能且呈剂量和时间依赖性;3β-HSD是该调节过程中的关键酶之一。

Objective Previous research discovered that after stimulation of GnRH on Leydig cells, the expression of annexin 5 and the level of testosterone were increased, then we guess that there may be interaction between annexin 5 and testosterone secretion. 3β-HSD is a marker enzyme in Leydig cells.This paper is to study the effect of annexin 5 on testosterone secretion and 3β-HSD expression in cultured rat Leydig cells. Methods The primary rat Leydig cells were cultured in vitro and treated with different concentration of annexin 5 （0,10-10,10-9,10-8mol/L） and with 10-9mol/L concentration of annexin 5 at varied time courses （6, 12, 24,36 h）.The culture media were collected for testosterone measurement by chemiluminescence assay. The expression of 3β-HSD protein were also analyzed by Western blot. Results The level of testosterone was significantly increased by 18%（14.38±0.30 vs 12.12±0.74）and 26%（15.25±0.47 vs 12.12±0.74）after treatment for 24 h with 10-10mol/L and 10-9mol/L concentration of annexin 5 （over control, P〈0.01） respectively. There is an increasing trend with treatment of annexin 5 （10-8mol/L）but no significant change（P〉0.05）; Western blotting results showed the 3β-HSD expression was significant increased by 63%（2.19±1.00 vs 1.34±0.19）（P〈0.01） at 10-9mol/L concentration but no significant change at 10-10and 10-8mol/L concentration. There was a correlation between the level of testosterone and 3β-HSD expression（r=0.430）. Compared with control group, after treatment with 10-9mol/L concentrations of annexin 5 for 24 h, the level of testosterone was significantly increased by 18%（12.59±0.41 vs 10.70±0.09）（P〈0.01）, and decreased by 21%（14.44±1.65 vs 17.46±0.31）（P〈0.01）for 36 h after stimulation. But there were no significant changes for 6h and 12 h. Conclusion In rat Leydig cells, annexin 5 regulates testosterone secretion in a dose-and time-dependent manner. 3β-HSD is one of key enzymes in the process of estosterone secretion.