摘要
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.
作者
王绍
周玥
向涵
刘星
沈炼桔
龙春兰
张德迎
何大维
林涛
魏光辉
Wang Shao;Zhou Yue;Xiang Han;Liu Xing;Shen Lianju;Long Chunlan;Zhang Deying;He Dawei;Lin Tao;Wei Guanghui(Department of Urology,Children’s Hospital of Chongqing Medical University/Chongqing Key Laboratory of Children Urogenital Development and Tissue Engineering/Ministry of Education Key Laboratory of Child Development and Disorders/China International Science and Technology Cooperation Base of Child Development and Critical Disorders/Chongqing Key Laboratory of Pediatrics)
出处
《重庆医科大学学报》
CAS
CSCD
北大核心
2019年第8期1003-1009,共7页
Journal of Chongqing Medical University
基金
国家自然科学基金面上资助项目（编号：81370782）
重庆医科大学附属儿童医院临床研究资助项目（编号：lcyj2015-5）
