雄激素受体干预对包皮成纤维细胞Mafb基因表达的影响

Effects of androgen receptor treatment on the expression of the Mafb gene in foreskin fibroblasts

目的:探讨双氢睾酮(dihydrotestosterone,DHT)与氟他胺(flutamide,Flu)对Mafb基因表达的影响。方法:利用原代培养的正常儿童包皮成纤维细胞株,分别采取不同浓度DHT与Flu干预,通过RT-PCR、免疫细胞荧光和Western blot检测Mafb的转录表达情况。结果:在DHT浓度为3.0×10^-8mol/L与3.0×10^-6mol/L干预条件下,Mafb mRNA的表达量分别为0.372±0.003、0.594±0.045,与正常组(0.444±0.007)相比,DHT 3.0×10^-6mol/L组明显上调(P=0.005),而DHT 3.0×10^-8mol/L组上调差异不明显(P=0.145);Flu 15μmol/L干预条件下,Mafb mRNA的表达量为0.221±0.013,与正常组相比,表达量明显下降(P=0.000)。细胞免疫荧光分析表明,与正常对照组相比(0.027±0.006),DHT 3.0×10^-8mol/L组(0.046±0.004)、3.0×10^-6mol/L组(0.076±0.003)Mafb表达量明显上调(P=0.010,P=0.000),而Flu组(0.006±0.002)明显下调(P=0.009)。同样Western blot结果显示,较对照组蛋白水平(0.163±0.001)相比,DHT 3.0×10^-8mol/L组(0.146±0.001)上调无统计学差异(P=0.960),DHT 3.0×10^-6mol/L组(0.598±0.087)Mafb表达量上调差异有统计学意义(P=0.000),而Flu组(0.066±0.002)明显下调(P=0.040)。结论:在体外培养包皮成纤维细胞株中,Mafb基因的表达受DHT和Flu的调控。Mafb作为雄激素受体下游调控蛋白,可能是雄激素受体信号通路促进尿道发育的重要机制。

Objective:To explore the effects of dihydrotestosterone(DHT) and flutamide(Flu) on the gene expression of Mafb.Methods:Primarily cultured normal foreskin fibroblasts from children were treated with different concentrations of DHT and Flu,and then were tested for the transcription and expression of Mafb using RT-PCR,cellular immunofluorescence,and Western blot. Results:After treatment with DHT at concentrations of 3.0×10^-8mol/L and 3.0×10^-6 mol/L,the mRNA expression of Mafb was significantly up-regulated in the DHT 3.0×10^-6 mol/L group(0.594±0.045 vs. 0.444±0.007,P=0.005),but not significantly up-regulated in the DHT 3.0×10^-8mol/L group(0.372±0.003 vs. 0.444±0.007,P=0.145),as compared with that in the normal group. After treatment with 15 μmol/L Flu,the mRNA expression of Mafb was significantly reduced compared with that in the normal group(0.221±0.013,P=0.000). Cellular immunofluorescence assay showed that the expression of Mafb was significantly up-regulated in the DHT3.0 ×10^-8mol/L group and the DHT 3.0 ×10^-6 mol/L group(0.046 ±0.004 and 0.076 ±0.003,respectively,P =0.010 and0.000,respectively),but significantly down-regulated in the Flu group(0.006±0.002,P=0.009),as compared with that in the normal control group(0.027±0.006). Similarly,Western blot results showed that the protein expression of Mafb was not significantly up-regulated in the DHT 3.0×10^-8mol/L group(0.146±0.001,P=0.960),but was significantly up-regulated in the DHT 3.0×10^-6 mol/L group(0.598±0.087,P=0.000),while the expression was significantly down-regulated in the Flu group(0.066±0.002,P=0.040),as compared with that in the control group(0.163±0.001). Conclusion:In the foreskin fibroblasts cultured in vitro,the gene expression of Mafb is regulated by DHT and Flu. Mafb,as a downstream regulatory protein of androgen receptor,may be an important mechanism in the androgen receptor signaling pathway to promote urinary tract development.