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目的:探讨肝脏17-α羟化酶(Cyp17A1)在糖异生中的作用。方法:采用实时定量PCR和蛋白印迹法检测正常C57BL/6小鼠在进食-饥饿-再进食状态下的肝脏糖异生关键酶[磷酸烯醇式丙酮酸羧激酶(phosphoenolpyruvate carboxykinase,PEPCK)、葡萄糖-6-磷酸酶(glucose-6-phosphatase,G6Pase)]基因及Cyp17A1的表达,比较正常小鼠与瘦素受体缺陷(db/db)小鼠肝脏中上述基因的表达差异;给予正常小鼠腹腔注射Cyp17A1下游产物17-羟孕酮(17-hydroxyprogesterone,17-OHP),并检测小鼠糖异生能力的改变和糖异生相关基因的表达;采用荧光素酶报告基因实验,检测17-OHP对糖皮质激素受体转录活性的调控作用。结果:在正常小鼠饥饿状态下及db/db小鼠中,肝脏糖异生关键酶基因(PEPCK及G6Pase)表达上调,肝脏Cyp17A1表达上调,其下游产物17-OHP含量升高;17-OHP处理组小鼠血糖升高,肝脏PEPCK及G6Pase mRNA水平显著增加;17-OHP通过激活糖皮质激素受体的转录活性,呈剂量依赖性上调糖异生关键酶基因(PEPCK、G6Pase)的mRNA水平。结论:肝脏Cyp17A1酶通过其下游产物17-OHP激活糖皮质激素受体转录活性,上调糖异生关键酶基因的m RNA水平,从而促进糖异生过程。
Objective: To investigate the role of liver 17-α-hydroxylase (Cyp17A1) in gluconeogenesis. Methods: Real - time quantitative PCR and Western blotting were used to detect the expression of hepatic gluconeo - pyruvate carboxykinase (PEPCK), glucose in normal C57BL / 6 mice under the condition of food intake, starvation and re - feeding 6-phosphatase (G6Pase)] gene and Cyp17A1 were detected by RT-PCR. The expression of these genes was compared between the normal mice and the leptin receptor deficient (db / db) The 17th-hydroxyprogesterone (17-OHP), a downstream product of Cyp17A1, was injected intraperitoneally. The changes of gluconeogenesis and the expression of gluconeogenesis related genes were detected in mice. Luciferase reporter assay was used to detect the expression of 17- OHP regulates the transcriptional activity of glucocorticoid receptor. Results: In normal mouse starvation and db / db mice, hepatic gluconeogenesis key enzyme genes (PEPCK and G6Pase) were up-regulated, hepatic Cyp17A1 expression was up-regulated, and 17-OHP content of hepatic products was increased. In the treatment group, the levels of PEPCK and G6Pase mRNA in liver increased significantly. 17-OHP increased the mRNA level of PEPCK (G6Pase) in a dose-dependent manner by activating the transcriptional activity of glucocorticoid receptor . CONCLUSION: The hepatic Cyp17A1 enzyme activates glucocorticoid receptor transcription activity through its downstream product 17-OHP, and increases the m RNA level of the key enzyme of gluconeogenesis, thereby promoting the gluconeogenesis process.