佛司可林对小鼠睾丸间质细胞雄激素合成酶及调节分子的影响

Effects of Forskolin on Androgen Synthetases and Regulatory Molecules in Mouse Leydig Cells

目的:研究佛司可林(FSK)对小鼠睾丸间质细胞雄激素合成的影响。方法:以TM3小鼠睾丸间质细胞为实验对象,分别采用1μmol/L、10μmol/L FSK处理TM3细胞15 min^24 h等不同时间,运用ELISA方法检测细胞分泌液睾酮含量,实时荧光定量PCR(RT-q PCR)方法检测细胞的雄激素合成相关基因mRNA表达。结果:与对照组比较,1μmol/L FSK处理24 h后对睾酮分泌无明显影响(P>0.05),但FSK可显著增强TM3细胞类固醇急性调节蛋白(Star)、胆固醇侧链裂解酶(Cyp11a1)基因表达上调(P<0.01);对17α羟化酶(Cyp17a1)、3β羟基类固醇脱氢酶(Hsd3b2)和黄体生成素受体(Lhr)基因表达无明显作用(P>0.05)。与对照组比较,采用10μmol/L FSK干预TM3细胞15 min^12 h,干预1 h起即可显著增强Star基因表达上调20倍以上(P<0.01);干预4 h起显著促进Cyp11a1基因表达上调4倍以上(P<0.05,P<0.01);FSK作用15 min起即可上调核受体Nr4a1基因表达(P<0.05),作用1 h时可显著增强Nr4a1基因表达上调达80倍(P<0.01);FSK干预1 h后可显著促进核受体Nr4a2基因表达上调达100倍以上(P<0.01);FSK作用8 h后才明显上调核受体Nr5a1基因表达(P<0.05)。结论:FSK能促进TM3小鼠睾丸间质细胞雄激素合成起始环节限速酶与即刻早期转录因子核受体基因转录,有助于雄激素合成前体物质孕烯醇酮以增加储备。

Objective: To study the effects of forskolin(FSK) on androgen synthesis in mouse leydig cells. Methods: The TM3 mouse leydig cells were observed and treated with FSK at concentrations of 1 μmol/L and 10 μmol/L for 15 minutes to 24 hours,respectively. The content of testosterone in cell secretion was detected by ELISA,and the mRNA expressions of genes related to androgen synthesis were detected by real time quantitative PCR(RT-q PCR). Results: Compared with the control group,there was no significant effect on the content of testosterone in cell secretion after treatment with FSK at concentration of1 μmol/L for 24 hours(P > 0. 05),but FSK could significantly up-regulate the mRNA expressions of steroid acute regulatory protein(Star) and cholesterol side chain cleavage enzyme(Cyp11a1) in TM3 cells(P < 0. 01),and there were no obvious effects on the mRNA expressions of 17α-hydroxylase(Cyp17a1) and 3β-hydroxysteroid dehydrogenase(Hsd3b2) and luteinizing hormone receptor(Lhr)(P > 0. 05). Compared with the control group,after treatment with FSK at concentration of10 μmol/L for 15 minutes to 12 hours,the mRNA expression of Star was significantly up-regulated over 20-fold after intervention for 1 hour(P < 0. 01). The mRNA expression of Cyp11a1 was significantly up-regulated over 4-fold after intervention for 4 hour(P < 0. 05,P < 0. 01). The mRNA expression of nuclear receptor Nr4a1 was up-regulated after FSK intervention for 15 minutes(P < 0. 05). After FSK intervention for 1 hours,the mRNA expression of Nr4a1 was significantly upregulated to 80-fold(P < 0. 01) and the mRNA expression of Nr4a2 was significantly up-regulated over 100-fold(P < 0. 01).The mRNA expression of Nr5a1 was obviously up-regulated after FSK intervention for 8 hours(P < 0. 05). Conclusion:Forskolin can promote the gene transcription of rate-limiting enzymes and immediate early transcription factors nuclear receptors in the start link of androgen synthesis in TM3 mouse leydig cells,which help to increase the reserve of pregnenolone as androgen synthesis precursors.