莱克多巴胺对人卵巢颗粒细胞类固醇激素合成急性调节蛋白表达的影响

Steroidogenic acute regulatory protein expression in human granulosa cells exposed to ractopamine in vitro

目的探讨β2肾上腺素受体(adrenergic receptor,AR)激动剂莱克多巴胺(ractopamine,RAC)对人卵巢颗粒细胞类固醇激素合成的效应。方法在人卵巢颗粒细胞体外培养系统中,添加高(50μmol/L)、中(5μmol/L)、低(0.5μmol/L)3种剂量的RAC,并设置空白对照(对照组)、50μmol/L克伦特罗(clenbuterol,CLB)和50μmol/L普萘洛尔组,在0 h、4 h、12 h、24 h和48 h回收细胞,采用实时荧光定量PCR和Western blotting检测颗粒细胞类固醇激素合成急性调节蛋白(steroidogenic acute regulatory protein,St AR)m RNA和蛋白表达水平的变化。结果各实验组颗粒细胞St AR m RNA的表达与其蛋白表达的变化趋势基本一致。对照组颗粒细胞各时间点St AR的表达水平差异无统计学意义(P>0.05)。CLB和RAC可升高St AR表达,普萘洛尔可抑制其表达。与0 h相比,St AR的表达水平在RAC作用后4 h达到高峰(P=0.000);12 h后,St AR的表达从峰值急剧下降(P=0.003);24 h后,基本恢复至基础水平(P>0.05)。不同剂量RAC与颗粒细胞共培养4 h后,St AR表达水平从高到低的顺序为:RAC低剂量组>RAC中剂组>RAC高剂量组,组间差异有统计学意义(P=0.000)。结论 RAC可升高颗粒细胞St AR的表达水平,影响卵巢类固醇激素合成功能,其后果可能引起女性生殖内分泌紊乱。

Objective To investigate the effects of ractopamine （RAC） on the expression of steroidogenic acute regulatory protein （STAR） in granulosa cells, aiming to illustrate the possible effects of RAC on steroidogenesis of the ovary. Methods Human granulosa cells were cultured in vitro with the additions of 0μmol/L RAC （control group）, 50μmol/L RAC, 5μmol/L RAC, 0.5μmol/L RAC, 50μmol/L clenbuteml （CLB） and 50μmol/L propranolol, respectively. After treatment, granulosa cells were harvested at 0 h, 4 h, 12 h, 24 h and 48 h. Protein and mRNA expressions of StAR were measured by Western blotting and real- time RT-PCR, respectively. Results After exposed to RAC, the changing tendency of StAR in mRNA level was similar to its protein level. StAR remained low level under control condition. RAC and CLB increased StAR expressions, and propranolol decreased its expression. StAR reached a peak after exposed to RAC for 4 h （P=0.000）, and underwent a sharp decline after 12 h treatment （P=0.003）. Thereafter, StAR restored to the basal level at 24 h （P〉0.05）. After coeultured with RAC for 4 h, 0.5 μmol/L RAC significantly increased StAR expression （P=0.000）. The expression of StAR in 5 μmol/L RAC group was intermediate to 0.5μmol/L RAC group and 50μmol/L RAC group. Conclusion RAC increased the expression of StAR in granulosa cells, which may enhance steroidgenesis of the ovary and result in the disturbance of steroid hormone homeostasis.