佛司可林促进小鼠肾上腺皮质瘤细胞皮质酮分泌的作用研究

摘要

Aim To study the effect of forskolin on cor-ticosterone secretion in mouse adrenocortical tumor cells. Methods Y1 cells were treated with 1μmol· L-1 or 10μmol·L-1 forskolin for 15 min to 24 h. Y1 cells growth morphology was observed, cell culture su-pernatants were collected and corticosterone was tested by ELISA kit. The cells total RNA was extracted using TRIzol kit and was reversely transcribed to obtain the cDNA, then was amplificated by real time quantitative PCR. The cells were lysed with RIPA lysis buffer and protein expressions were carried out by Western blot. Results Y1 cells morphology changed from flat adher-ent to shrink and spherical growth after 1μmol · L-1 forskolin treatment for 3 h. Compared with the control group, corticosterone levels were increased significantly by 1μmol · L-1 forskolin treatment for 24 h ( P <0. 01), then, forskolin significantly enhanced steroid ogenic acute regulatory protein ( Star) mRNA and pro-tein expression ( P <0. 01 ) , moreover, the steroido-genic enzymes such as Cyp11 a1 and Cyp11 b1 mRNA expressions were also up-regulated significantly by fors-kolin ( P <0. 01 ) . Additionally, Star mRNA expres-sion was increased significantly in a time-dependent re-sponse after 10μmol·L-1 forskolin treatment from 1 h to 12 h (P<0. 01). Furthermore, Nr4a1 and Nr4a2 mRNA expressions were up-regulated significantly after 10μmol · L-1 forskolin treatment from 15 min and reached the top at 1 h ( P<0. 01 ) . However, forsko-lin showed no effect on Mc2 r and Nr5 a1 mRNA expres-sions. Conclusion Y1 mouse adrenocortical tumor cells have a strong response to adenylate cyclase ago-nists, then, forskolin can be used to glucocorticoid se-cretion inducer reagent.%目的：研究佛司可林( FSK)对小鼠肾上腺皮质瘤细胞皮质激素的促分泌作用。方法以Y1小鼠肾上腺皮质瘤细胞为实验对象,采用1、10μmol·L-1 FSK处理Y1细胞15 min~24 h等不同时程,观察 Y1细胞生长形态变化,运用ELISA方法检测细胞分泌液皮质酮含量,RT-qPCR方法检测相关基因mRNA表达,Western blot方法检测相关蛋白表达。结果1μmol·L-1 FSK干预Y1细胞3 h后细胞形态开始皱缩并逐渐形成圆球状生长。与对照组比较,1μmol · L-1 FSK治疗24 h后明显升高Y1细胞皮质酮水平( P<0．01)；并增加类固醇急性调节蛋白酶( Star)基因和蛋白表达( P<0．01)、也明显升高皮质酮合成第一步骤限速酶Cyp11a1和皮质酮合成最后一步关键酶Cyp11b1基因表达(P<0．01)。此外,10μmol·L-1 FSK干预Y1细胞15 min~12 h不同时程后,发现1 h后即可明显增加Star基因表达,并呈现典型的时效关系(P<0．01)；孤儿核受体(Nr4a1和Nr4a2)基因表达在FSK处理15 min后明显升高、1 h后上调最明显( P<0．01),之后升高幅度逐渐减弱。然而,FSK对孤儿核受体(Nr5a1)和促肾上腺皮质激素受体(Mc2r)基因表达无明显影响。结论 Y1小鼠肾上腺皮质瘤细胞对腺苷酸环化酶激活剂佛司可林具有强烈的应答反应,佛司可林是皮质激素分泌的强有效诱导剂。