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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(P<0.05);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(P<0.05)。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。
OBJECTIVE: To observe the effect of Ras homolog in rheumatoid factor of rapamycin (mTOR) signaling on the differentiation of 3T3-L1 cells. Methods: 3T3-L1 preadipocyte line was transfected with gene recombinant plasmid with high expression of Rheb. Western blotting experiments identified the successful transfection of plasmids and induced differentiation of the cells. The 3T3-L1 cells were oily red stained on the 8th day of differentiation, and the content of intracellular triglyceride was detected. In addition, we examined the expression of peroxisome proliferator-activated receptor-γ (PPAR-γ) and C / EBP-α (CCAAT-enhancer-binding protein-α) The role of cell differentiation process. Results: We successfully constructed the 3T3-L1 cell line with high expression of Rheb, and found that the lipid droplets can be promoted by the high expression of Rheb, the oil red O staining is significantly different. Compared with the control group, the triglyceride content of Rheb high expression group (P <0.05). The expression of adipocyte-specific transcription factors such as C / EBP-α and PPAR-γ also increased compared with the control group (P <0.05). Conclusion: Rheb gene, as upstream regulator of mTOR pathway, can promote adipocyte differentiation.