mTOR信号通路中上游调控蛋白Rheb在脂肪细胞分化中的作用

Role of a regulating protein in the upper stream of mTOR signaling pathway Rheb in adipocyte differentiation

目的观察mTOR信号通路中上游调控蛋白Rheb对脂肪细胞分化的影响。方法构建Rheb(Ras homologenriched in brain)的重组质粒(pCAG-Insulator-Rheb),并将其注射到B6小鼠的胚胎内,产生全身高表达Rheb的转基因小鼠。提取怀孕雌鼠第13.5天的胚胎成纤维细胞(mouse embryonic fibroblast cells,MEFs),PCR鉴定基因型之后进行成脂诱导分化。通过检测分化第12天MEFs内三酰甘油的含量、油红O染色的结果、实时定量PCR方法检测脂肪细胞特异性转录因子PPARγ和C/EBPα的表达情况,来观察Rheb在脂肪细胞分化过程中的作用。结果成功构建了全身高表达Rheb的转基因小鼠模型,并且检测到在高表达Rheb后,可以促进MEFs内脂滴的生成,三酰甘油含量增高,油红O染色有明显区别,并且脂肪细胞特异性转录因子PPARγ和C/EBPα的表达量均升高。结论 Rheb过表达以后可以促进脂肪细胞的分化。

Objective To observe the effect of Rheb, a regulating protein in the upper stream of mTOR signaling pathway, on adipocyte differentiation. Methods （1） The recombinant plasmid pCAG-Insulator-Rheb was constructed and injected into the embryo of B6 mouse to produce transgenic mouse over-expressing Rheb. （2） The mouse embryonic fibroblast cells （MEFs） were collected from Rheb transgenic mice on pregnancy day 13. 5, and they were induced to differentiate into adipoeytes after identified by PCR. （3） On differentiation day 12, the MEFs were subjected to Oil Red O staining and the contents of triglyceride in the MEFs were determined; moreover, the expression of special transcription factor PPAR7 and C/ EBP~ was examined by real-time PCR, and all of the above parameters were used to assess the role of Rheb in the differentiation of adipoeytes. Results We successfully constructed the transgenic mouse model over-expressing Rheb. We found that mouse over-expressing Rheb promoted adipogenesis in MEFs and increased the content of triglyeeride in MEFs; moreover, the overexpression also greatly changed the results of Oil Red O staining and greatly promoted the expression of adipocyte specific transcript factor PPARγ and C/EBPα. Conclusion Over-expression of Rheb can promote the differentiation of MEFs into adipocytes.