摘要
目的观察Lim家族成员Ajuba对糖异生关键基因转录水平的影响,探讨Ajuba参与肝糖代谢调节的分子机制。方法 1慢病毒感染人肝癌细胞系Hep G2,筛选低表达和过表达Ajuba的稳转细胞,通过Western blotting鉴定Ajuba表达情况。2real-time PCR检测Hep G2稳转细胞中糖异生关键酶磷酸烯醇式丙酮酸羧激酶(PEPCK)和葡萄糖-6-磷酸酶(G6P)基因mRNA表达。3分离培养C57BL/6小鼠原代肝细胞并以慢病毒感染法筛选低表达Ajuba的细胞,real-time PCR检测Pepck和G6p mRNA水平。结果 1Western blotting结果显示,低表达和高表达Ajuba的Hep G2细胞均构建成功。2与对照组相比,低表达Ajuba的Hep G2细胞中PEPCK和G6P mRNA水平明显降低,而过表达细胞中则显著升高(P<0.05)。3低表达Ajuba的小鼠原代肝细胞中Pepck和G6p mRNA水平明显降低(P<0.05)。结论 Lim蛋白Ajuba能够通过促进肝糖异生关键基因PEPCK和G6P的表达参与糖代谢调节,且该作用不具有种属特异性。
Objective To observe the effects of Lim protein Ajuba on transcriptional levels of key genes relevant to gluconeogenesis and explore the molecular mechanism of regulating the liver glycogen metabolism by Ajuba. Methods ①Human hepatoma cells HepG2 were infected with lentivirus and stable transfected cells with low/ high expression of Ajuba were screened. The expression of Ajuba was detected by the Western blotting. ②Expressions of two key enzymes relevant to gluconeogenesis, i.e. phosphoenolpyruvate carboxykinase （PEPCK） and glucose-6-phosphatase （G6P）, were detected by the real-time PCR. ③Primary hepatocytes of C57BL/6 mice were isolated and cells with low expression of Ajuba were screened by the lentivirus infection method. The mRNA expressions of Pepck and G6p were detected by the real-time PCR. Results ①The results of Western blotting showed that HepG2 cells with low and high expressions of Ajuba were successfully screened. ②Compared with controls, mRNA levels of PEPCK and G6P in HepG2 cells with low expression of Ajuba significantly decreased, while mRNA levels of PEPCK and G6P in HepG2 cells with high expression of Ajuba significantly increased （P 〈0.05）. ③The mRNA levels of Pepck and G6p in mouse primary hepatocytes with low expression of Ajuba significantly decreased （P 〈 0.05）. Conclusion The Lim protein Ajuba can participate in the regulation of glucose metabolism by up-regulating the expressions of PEPCK and G6P, two key genes relevant to gluconeogenesis. This effect is not species-specific.
出处
《上海交通大学学报（医学版）》
CAS
CSCD
北大核心
2015年第5期637-641,共5页
Journal of Shanghai Jiao tong University:Medical Science
基金
国家自然科学基金项目(81372028)
上海市科委医学引导项目(124119a5700)~~
