类固醇生成因子-1基因沉默在介导血管紧张素Ⅱ对醛固酮分泌调控的影响

Influences of steroidogenic factor-1 gene silencing on angiotensin Ⅱ -mediated aldosterone secretion

目的 观察血管紧张素Ⅱ（ATⅡ）刺激人肾上腺皮质癌H295R细胞后醛固酮合成酶（CYP11B2）mRNA及醛固酮激素的改变,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 用携带U6启动子和SF-1特异性短发夹RNA（shRNA）序列的质粒载体pGenesil1-SF-1-shRNA及含非特异性shRNA编码序列的阴性对照质粒pGenesill-negative-shRNA转染人肾上腺皮质癌H295R细胞,应用Westem blot和荧光定量聚合酶链反应（PCR）法检测SF-1表达的改变,以100 nmol/L浓度ATⅡ刺激实验组和对照组细胞,检测ATⅡ刺激后3、6、9、12、15、18 h时间点CYP11B2 mRNA表达水平,与未刺激时对比,检测两组细胞CYP11B2 mRNA的最大增幅.用放射免疫法测定醛固酮激素在ATⅡ刺激24 h后的分泌水平,对比两组细胞醛固酮增幅.结果 实验组细胞SF-1在蛋白水平和mRNA水平分别下降了69.07%和71.20%（P＜0.01）.ATⅡ刺激后12 h时CYP11B2 mRNA表达水平最高,与未刺激前比较,实验组和对照组的CYP11B2 mRNA增幅分别是50.21倍和800.09倍（P＜0.01）,后者是前者的15.93倍（P＜0.01）.同样,ATⅡ刺激后醛固酮激素分泌水平均升高,对照组细胞刺激前后分别为（0.061±0.007）μg/L和（0.256±0.014）μg/L（P＜0.01） 而实验组醛固酮激素在ATⅡ刺激前后则分别为（0.101±0.010）μg/L和（0.252±0.016）μg/L（P＜0.01）,两组比较,前者增幅高于后者1.7倍（P＜0.01）.结论 SF-1表达下调可以降低CYP11B2 mRNA及醛固酮对ATⅡ的敏感性和反应性.

Objective To observe the changes of CYP11B2 mRNA and aldosterone secretion in human adrenocortical carcinoma H295R cells after angiotensin Ⅱ （AT Ⅱ ） stimulation, and investigate the effects of steroidogenic factor-1 （ SF-1 ） gene silencing. Methods The plasmids pGenesil1-SF-1-shRNA containing U6 promoter and SF-1 -specific short hairpin RNA （shRNA） or pGenesil1 -negative-shRNA containing unspecific shRNA were transfected into the H295R cells. The expression of SF-1 was detected by Western blotting and real-time polymerase chain reaction （RT-PCR）. CYP11B2 mRNA was detected at 3,6, 9, 12, 15, 18 h after ATⅡ stimulation （ 100 nmol/L）, and the maximal amplitude was compared between two groups. Aldosterone was measured by radioimmunoassay at 3, 6, 9, 12, 15, 18 h after AT Ⅱ stimulation （ 100 nmol/L）, and the amplitude of aldosterone was compared between two groups. Results As compared with those in control group, the protein and mRNA levels in SF-1-transfected group were reduced by 69.07% and 71.20% respectively. The highest CYP11B2 mRNA level was detected at 12 h after ATⅡ stimulation, and the amplitude in SF-1-transfected group and non-transfection group was 50. 21 fold and 800.09 fold respectively compared to that of cells without ATⅡ stimulation （ P 〈 0.01 ）. Similarly, aldosterone secretion in control group was （0. 061 ± 0. 007 ） and （0. 256 ± 0.014） μg/L before and after ATⅡ intervention （P〈0. 01）, and that in SF-1-transfected group was （0. 101 ±0.010） and （0.252 ±0.016） μg/L （P 〈 0. 01 ） respectively. Conclusion Down-regulation of SF-1 decreased the sensitivity and response of CYP11B2 mRNA and aldosterone to ATⅡ stimulation.