CAJ | 학술논문

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.
목적 관찰촉신상선피질격소(ACTH)자격인신상선피질암H295R세포후대철고동화피질순적조공영향,병탐토류고순생성인자-1(SF-1)기인침묵재개도해반응중적작용.방법 용만병독포장휴대SF-1특이성단발협RNA(shRNA)서렬적재체pLVX-shRNA-SF-1,동시설립음성급공백대조조,전염인신상선피질암H295R세포,전염후48 h응용Western blot화실시정량취합매련반응(Real-time PCR)법검측SF-1표체수평.설치100 nmol/L ACTH자격실험조화대조조세포,검측자격1h후류고순급성조절단백(StAR) mRNA표체수평,검측자격48 h후철고동합성매CYP11B2화피질순합성매CYP11B1 mRNA표체수평;채용매련면역흡부시험(ELISA)측정동시기철고동화피질순격소적함량,비교량조세포철고동화피질순증폭.결과 실험조세포SF-1재단백수평화mRNA수평분별하강료69％화71％ (P＜0.05).ACTH자격1h시실험조비대조조StARmRNA표체수평승고1.4배,량조세포철고동수평분별위(91.59 ±4.28)화(79.90±2.41) ng/L(P＜0.05),단피질순수평분별위(23.72±1.50)화(23.74±1.40) ng/L(P ＞0.05).자격48 h시실험조CYP11B2기인표체수평시대조조세포적13배,단철고동표체함량분별위(129.50 ±6.87)、(131.52 ±7.56) ng/L(P＞0.05);실험조CYB11 B1 mRNA표체수평부위대조조세포적1.18배(P＞0.05),단피질순함량위(28.95 ±1.51)화(33.08±1.67) ng/L(P＜0.05).량충격소재ACTH자격후대비각자증폭,대조조분별위실험조적1.37배화1.80배(P＜0.05).결론 SF-1표체하조가이강저철고동화피질순대ACTH적민감성화반응성.
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 higher than that of transfection group (P ＜ 0.05).Conclusion Down-regulation of SF-1 decreases the sensitivity and response of aldosterone and cortisol to ACTH stimulation.