Central Laboratory, Beijing Shijitan Hospital of Capital Medical University, Beijing 100038, P.R. China.
Mol Med Rep. 2018 Apr;17(4):6109-6115. doi: 10.3892/mmr.2018.8619. Epub 2018 Feb 16.
Primary adipose-derived stem cells (ADSCs) are a mixture of cell types including preadipocytes having the ability to spontaneously differentiate into adipocytes. The aim of the present study was to compare the spontaneous adipogenic differentiation potential of ADSCs at different passages to determine whether it decreased with continuous cell passages. Mouse ADSCs (mADSCs) were harvested and cells from passages 1 to 5 were used for experiments. The proliferation of mADSCs at different passages was tested using the cell counting kit‑8 assay. Reverse transcription‑quantitative polymerase chain reaction was used to determine relative mRNA expression levels of CCAAT/enhancer binding protein α (Cebpa and C/EBPα) and peroxisome proliferator‑activated receptor γ (Pparg and PPARγ), and western blot analysis was used to investigate C/EBPα and PPARγ protein expression. The cells were cultured using DMEM. The fixed cells were then stained using Oil Red O on days 14 and 28, and the obtained extracted dye was monitored for absorbance. The 510 nm absorbance from passages 1 to 5 was observed to be statistically different. The relative expression levels of Cebpa and Pparg for mADSCs from passage 1 were significantly higher when compared with those for mADSCs from passages 2 to 5 on days 3, 5 and 7. However, no difference was identified in the expression levels of proteins C/EBPα and PPARγ for different passages. Although the mRNA expression levels of Pparg from passages 4 to 5 were significantly higher when compared with those from passage 1, the results of Oil Red O absorbance, mRNA expression levels of Cebpa, and the protein expression levels of C/EBPα and PPARγ exhibited no difference between mADSCs from passages 1 to 5 when cultured with induced adipogenic differentiation medium. Therefore, it was concluded that the spontaneous adipogenic differentiation potential of ADSCs decreased with continuous cell passages.
原代脂肪来源干细胞（ADSCs）是一种细胞类型的混合物，包括具有自发分化为脂肪细胞能力的前脂肪细胞。本研究旨在比较 ADSC 在不同传代中的自发脂肪分化潜能，以确定其是否随细胞连续传代而降低。从小鼠 ADSC（mADSC）中分离细胞，并用于实验的传代 1 至 5。使用细胞计数试剂盒-8 测定不同传代中 mADSC 的增殖。采用逆转录-定量聚合酶链反应测定 CCAAT/增强子结合蛋白α（Cebpa 和 C/EBPα）和过氧化物酶体增殖物激活受体γ（Pparg 和 PPARγ）的相对 mRNA 表达水平，采用 Western blot 分析检测 C/EBPα 和 PPARγ 蛋白表达。使用 DMEM 培养细胞。第 14 天和第 28 天，用油红 O 固定细胞染色，监测获得的提取染料的吸光度。观察到传代 1 至 5 的 510nm 吸光度具有统计学差异。与传代 2 至 5 相比，第 3、5 和 7 天，传代 1 的 mADSC 的 Cebpa 和 Pparg 的相对表达水平显著更高。然而，不同传代的 C/EBPα 和 PPARγ 蛋白表达水平没有差异。虽然与传代 1 相比，传代 4 和 5 的 Pparg 的 mRNA 表达水平显著更高，但在诱导脂肪分化培养基中培养时，mADSC 从传代 1 至 5 的油红 O 吸光度、Cebpa 的 mRNA 表达水平以及 C/EBPα 和 PPARγ 的蛋白表达水平没有差异。因此，结论是 ADSC 的自发脂肪分化潜能随细胞连续传代而降低。
