Department of Regenerative Medicine, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran.
Cell Biol Int. 2011 Jul;35(7):713-20. doi: 10.1042/CBI20100457.
HSPs (heat shock proteins) have been recognized to maintain cellular homoeostasis during changes in microenvironment. The present study aimed to investigate the HSPs expression pattern in hierarchical neural differentiation stages from mouse embryonal carcinoma stem cells (P19) and its role in heat stressed exposed cells. For induction of HSPs, cells were heated at 42°C for 30 min and recovered at 37°C in different time points. For neural differentiation, EBs (embryoid bodies) were formed by plating P19 cells in bacterial dishes in the presence of 1 mM RA (retinoic acid) and 5% FBS (fetal bovine serum). Then, on the sixth day, EBs were trypsinized and plated in differentiation medium containing neurobasal medium, B27, N2 and 5% FBS and for an extra 4 days. The expression of HSPs and neural cell markers were evaluated by Western blot, flow cytometry and immunocytochemistry in different stages. Our results indicate that HSC (heat shock constant)70 and HSP60 expressions decreased following RA treatment, EB formation and in mature neural cells derived from heat-stressed single cells and not heat-treated EBs. While the level of HSP90 increased six times following maturation process, HSP25 was expressed constantly during neural differentiation; however, its level was enhanced with heat stress. Accordingly, heat shock 12 h before the initiation of differentiation did not affect the expression of neuroectodermal and neural markers, nestin and β-tubulin III, respectively. However, both markers increased when heat shock was induced after treatment and when EBs were formed. In conclusion, our results raise the possibility that HSPs could regulate cell differentiation and proliferation under both physiological and pathological conditions.
热休克蛋白（HSPs）被认为在微环境变化时维持细胞内稳态。本研究旨在探讨从小鼠胚胎癌细胞干细胞（P19）的层次神经分化阶段 HSPs 的表达模式及其在受热应激暴露细胞中的作用。为了诱导 HSPs，将细胞在 42°C 下加热 30 分钟，然后在不同时间点恢复到 37°C。为了进行神经分化，通过在存在 1mM RA（视黄酸）和 5％FBS（胎牛血清）的细菌培养皿中接种 P19 细胞来形成 EBs（胚状体）。然后，在第六天，将 EBs 用胰蛋白酶消化并接种在含有神经基础培养基、B27、N2 和 5％FBS 的分化培养基中，再培养 4 天。在不同阶段通过 Western blot、流式细胞术和免疫细胞化学评估 HSPs 和神经细胞标志物的表达。我们的结果表明，在 RA 处理、EB 形成以及从受热应激单细胞和未受热处理的 EBs 衍生的成熟神经细胞中，HSC（热休克常数）70 和 HSP60 的表达降低。而 HSP90 的水平在成熟过程中增加了六倍，HSP25 在神经分化过程中持续表达；然而，其水平在受热应激时增强。因此，在分化开始前 12 小时进行热休克不会影响神经外胚层和神经标记物巢蛋白和β-微管蛋白 III 的表达。然而，当诱导热休克后进行处理和形成 EBs 时，这两种标记物的表达均增加。总之，我们的结果提出了 HSPs 可能在生理和病理条件下调节细胞分化和增殖的可能性。
