Rheb过表达在前体脂肪细胞株3T3-L1分化中的作用

目的:利用前体脂肪细胞株3T3-L1细胞观察mTOR(mammalian target of rapamycin)信号通路中上游调控因子Rheb(Ras homolog enriched in brain)对其分化的影响。方法:利用高表达Rheb的基因重组质粒转染前体脂肪细胞株,3T3-L1。通过蛋白质免疫印迹实验鉴定质粒成功转染细胞后,诱导该细胞脂肪分化。予以分化第8天的3T3-L1细胞油红染色,并检测细胞内甘油三酯的含量。另外,我们用Western blot方法检测脂肪细胞特异性转录因子PPAR-γ(Peroxisome proliferator-activated receptor-γ)和C/EBP-α(CCAAT-enhancer-binding protein-α)的表达情况来研究Rheb在脂肪细胞分化过程中的作用。结果:我们成功构建了高表达Rheb的3T3-L1细胞株,发现高表达Rheb后可以促进脂滴的生成,油红O染色有显著区别,与对照组相比Rheb高表达组的三酰甘油含量明显升高(P0.05);C/EBP-α和PPAR-γ等脂肪细胞特异性的转录因子蛋白表达量与对照组相比也均有升高(P0.05)。结论:Rheb基因作为mTOR通路上游调控因子,可以促进脂肪细胞的分化。

The Function of Rheb Overexpression in 3T3-L1 Pre-adipocyte Differentiation

Objective:To observe the effects of Rheb, an upstream regulating factor in mTOR (mammalian target of rapamycin) signaling pathway, on the differentiation of pre-adipocytes, 3T3-L1.Methods:The plasmid pCAG-Insulator-Rheb was transfected into mouse pre-adipocyte 3T3-L1 cells to overexpress Rheb. Western blot analysis was applied to verify the overexpression in 3T3-L1 cells. On day 8 of differentiation, the 3T3-L1cells were stained with Oil Red O dye and analyzed for triglyceride contents in the 3T3-L1 cells. Moreover, the expression of adipocyte differentiation marker genes, PPAR-r(Peroxisome proliferator-activated receptor-r) and C/EBP-alpha (CCAAT-enhancer-binding protein-alpha), were examined by Western blot analysis. These parameters were used to assess the role of Rheb in the differentiation of 3T3-L1 cells.Results:We successfully overexpressed Rheb in 3T3-L1 cells. Overexpressing Rheb promoted adipogenesis and increased the content of triglyceride (P<0.05) judging by the elevated Oil Red O staining and expression of adipocyte marker genes, PPAR-r and C/EBP-alpha(P<0.05).Conclusion:As the upstream regulatory factor of mTOR pathway, Rheb can promote adipocyte differentiation.