类固醇生成因子-1表达下调在介导促肾上腺皮质激素对H295R细胞醛固酮和皮质醇急慢性分泌调控中的作用  

Regulatory effect of steroidogenic factor-1 gene silencing on aldosterone and cortisol secretion to adreno-cortico-tropic-hormone stimulation in H295R cells during acute and chronic phases

作　　者：胡东亮[1] 丁协刚[1] 曹锐[1] 倪栋[1] 刘同族[1] 王行环[1] 

机构地区：[1]武汉大学中南医院泌尿外科,430071

出　　处：《中华实验外科杂志》2014年第12期2703-2705,共3页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金资助项目（81200579）

摘　　要：目的 观察促肾上腺皮质激素（ACTH）刺激人肾上腺皮质癌H295R细胞后对醛固酮和皮质醇的调控影响,并探讨类固醇生成因子-1（SF-1）基因沉默在介导该反应中的作用.方法 用慢病毒包装携带SF-1特异性短发夹RNA（shRNA）序列的载体pLVX-shRNA-SF-1,同时设立阴性及空白对照组,转染人肾上腺皮质癌H295R细胞,转染后48 h应用Western blot和实时定量聚合酶链反应（Real-time PCR）法检测SF-1表达水平.设置100 nmol/L ACTH刺激实验组和对照组细胞,检测刺激1h后类固醇急性调节蛋白（StAR） mRNA表达水平,检测刺激48 h后醛固酮合成酶CYP11B2和皮质醇合成酶CYP11B1 mRNA表达水平;采用酶联免疫吸附试验（ELISA）测定同时期醛固酮和皮质醇激素的含量,比较两组细胞醛固酮和皮质醇增幅.结果 实验组细胞SF-1在蛋白水平和mRNA水平分别下降了69％和71％ （P＜0.05）.ACTH刺激1h时实验组比对照组StARmRNA表达水平升高1.4倍,两组细胞醛固酮水平分别为（91.59 ±4.28）和（79.90±2.41） ng/L（P＜0.05）,但皮质醇水平分别为（23.72±1.50）和（23.74±1.40） ng/L（P ＞0.05）.刺激48 h时实验组CYP11B2基因表达水平是对照组细胞的13倍,但醛固酮表达含量分别为（129.50 ±6.87）、（131.52 ±7.56） ng/L（P＞0.05）;实验组CYB11 B1 mRNA表达水平仅为对照组细胞的1.18倍（P＞0.05）,但皮质醇含量为（28.95 ±1.51）和（33.08±1.67） ng/L（P＜0.05）.两种激素在ACTH刺激后对比各自增幅,对照组分别为实验组的1.37倍和1.80倍（P＜0.05）.结论 SF-1表达下调可以降低醛固酮和皮质醇对ACTH的敏感性和反应性.Objective To study the changes in aldosterone and cortisol secretion in human adrenocortical carcinoma H295R cells after adreno-cortico-tropic-hormone （ACTH） stimulation,and investigate the effect of steroidogenic factor-1 （SF-1） gene silencing on them.Methods Lentiviral vector pLVX-specific short hairpin RNA （shRNA）-SF-1 containing SF-1-specific shRNA was transfected into the H295R cells,and negative-control and black groups were set up simuhaneously.SF-1 expression was detected by Western blotting and real-time quantitative polymerase chain reaction （Real-time PCR） at 48 h after transfection.Steroidogenic acute regulatory protein （StAR） mRNA was detected at 1 h after ACTH stimulation （100 nmol/L）,and CYB1 1 B2/B1 mRNA was detected at 48 h after ACTH stimulation （100 nmol/L）.Aldosterone and cortisol were measured by enzyme linked immunosorbent assay （ELISA） analysis,and the increase amplitude was compared.Results As compared with those in control group,the protein and mRNA levels in pLVX-shRNA-SF-1-transfected cells were reduced by 69% and 71% respectively.StAR mRNA in transfection group was 1.4-fold higher than that of the control group at 1 h after ACTH stimulation （P 〈 0.05）.Similarly,the aldosterone production in two groups was （91.59-± 4.28） and （79.90 ± 2.41） ng/L （P〈0.05）,and cortisol production was （23.72 ± 1.50） and （23.74 ± 1.40） ng/L （P〉0.05）,respectively.A 13-fold increase in CYP1 1 B2 mRNA levels was observed in transfection group as compared with the control group at 48 h after ACTH stimulation （P 〈0.05）.However,aldosterone levels in two groups were （129.50±6.87） and （131.52 ± 7.56） ng/L respectively （P 〉 0.05）.CYP11B1 mRNA was 1.18-fold higher in transfection group than that of the control group （P 〉 0.05）,and cortisol production was （28.95 ± 1.51） and （33.08 ± 1.67） ng/L respectively （P 〈 0.05）.For aldosterone and cortisol,the amplification in control group was 1.37 and 1.80 times hig

关 键 词：类固醇生成因子-1 肾上腺皮质癌 促肾上腺皮质激素 醛固酮 皮质醇 

分 类 号：R58[医药卫生—内分泌]