Laboratory of Stem Cell and Tumor Biology, College of Veterinary Medicine, Seoul National University, Seoul, Korea.
Cell Prolif. 2010 Feb;43(1):19-28. doi: 10.1111/j.1365-2184.2009.00650.x.
In recent years, obesity has become a global epidemic, highlighting the necessity for basic research into mechanisms underlying growth of adipose tissue and differentiation of stem cells into adipocytes, in humans. For better understanding of cell signalling in adipogenesis, the role of DNER (delta/Notch-like EGF-related receptor) in adipogenic differentiation of human adipose tissue-derived mesenchymal stem cells (hAMSC) was investigated.
To assess the role of DNER in hAMSC adipogenesis, hAMSCs were transfected with DNER small interfering RNA (siDNER). Real-time quantitative reverse transcriptase polymerase chain reactions to assess expression levels of adipogenesis-related genes regulated by siDNER, cell cycle and immunoblot analyses were performed.
First, it was determined that DNER mRNA was profoundly expressed in hAMSCs and reduced during adipogenic differentiation. Knockdown of DNER altered cell morphology, inhibited proliferation and increased frequency and efficiency of adipogenesis in hAMSC. Expression of CCAAT/enhancer-binding protein delta increased and proportion of cells in S phase decreased by knockdown of DNER, using specific siRNA. Moreover, adipocyte-specific genes including peroxisome proliferator-activated receptor gamma, fatty acid binding protein 4 and perilipin were up-regulated in siDNER compared to the siControl group during adipogenesis in hAMSC.
These results indicate that DNER knockdown in hAMSC accelerated onset of adipogenic differentiation by bypassing mitotic clonal expansion during the early stages of adipogenesis.
近年来，肥胖已成为全球性问题，突显了对脂肪组织生长和干细胞向脂肪细胞分化的机制进行基础研究的必要性。为了更好地了解脂肪生成中的细胞信号转导，研究了 DNER（delta/Notch 样表皮生长因子受体）在人脂肪组织来源间充质干细胞（hAMSC）的脂肪生成分化中的作用。
为了评估 DNER 在 hAMSC 脂肪生成中的作用，用 DNER 小干扰 RNA（siDNER）转染 hAMSCs。通过实时定量逆转录聚合酶链反应评估受 siDNER 调控的脂肪生成相关基因的表达水平、细胞周期和免疫印迹分析。
首先确定 DNER mRNA 在 hAMSCs 中高度表达，并在脂肪生成分化过程中减少。DNER 的敲低改变了细胞形态，抑制了增殖，并增加了 hAMSC 中脂肪生成的频率和效率。用特异性 siRNA 敲低 DNER 后，CCAAT/增强子结合蛋白 δ 的表达增加，S 期细胞的比例减少。此外，在 hAMSC 的脂肪生成过程中，与 siControl 组相比，脂肪细胞特异性基因包括过氧化物酶体增殖物激活受体 γ、脂肪酸结合蛋白 4 和 perilipin 在 siDNER 中上调。
这些结果表明，在 hAMSC 中敲低 DNER 通过在脂肪生成的早期阶段绕过有丝分裂克隆扩增，加速了脂肪生成分化的开始。
