Laboratoire Réponses cellulaires et fonctionnelles à l'hypoxie, Université Paris 13, Bobigny, France.
Cell Prolif. 2010 Feb;43(1):1-8. doi: 10.1111/j.1365-2184.2009.00648.x.
The aim of this study was to evaluate whether hypoxia and/or erythropoietin would be able to modulate proliferation/differentiation processes of rat and human myoblasts.
Rat L6 and primary human myoblasts were grown in 21% or 1% O(2) in the presence or absence of recombinant human erythropoietin (RhEpo). Presence of erythropoietin receptors (EpoR) was assayed using RT-PCR and Western blotting techniques. Cell proliferation was evaluated by determining the doubling time and kinetics of cultures by counting cells. Cell differentiation was analysed by determining myogenic fusion index using antibodies against the myosin heavy chain. Expression of myogenin and myosin heavy chain (MHC) proteins were evaluated using the Western blotting technique.
After 96 h culture in growth medium for 2.5 and 9 h, doubling time of L6 and human primary myoblasts respectively, had increased in 1% O(2) conditions (P < 0.01). Kinetics of culture showed alteration in proliferation at 72 h in L6 myoblast cultures and at 4 days in human primary myoblasts. The myogenic fusion index had reduced by 30% in L6 myoblasts and by 20% in human myoblasts (P < 0.01). Expression of myogenin and MHC had reduced by around 50%. Despite presence of EpoR mRNA and protein, RhEpo did not counteract the effects of hypoxia either in L6 cells or in human myoblasts.
The data show that exposure to hypoxic conditions (1% O(2)) of rat and human myoblasts altered their proliferation and differentiation processes. They also show that Epo is not an efficient growth factor to counteract this deleterious effect.
本研究旨在评估低氧和/或促红细胞生成素是否能够调节大鼠和人肌母细胞的增殖/分化过程。
将大鼠 L6 细胞和原代人肌母细胞分别在 21%或 1% O2 下培养，同时存在或不存在重组人促红细胞生成素（rhEpo）。使用 RT-PCR 和 Western blot 技术检测红细胞生成素受体（EpoR）的存在。通过细胞计数来确定倍增时间和培养动力学来评估细胞增殖。通过用肌球蛋白重链抗体测定肌生成融合指数来分析细胞分化。使用 Western blot 技术评估肌生成素和肌球蛋白重链（MHC）蛋白的表达。
在生长培养基中培养 96 小时后，L6 和人原代肌母细胞的倍增时间分别增加了 1% O2 条件下（P < 0.01）。培养动力学显示 L6 肌母细胞培养物在 72 小时和人原代肌母细胞在 4 天的增殖发生变化。L6 肌母细胞的肌生成融合指数降低了 30%，人肌母细胞降低了 20%（P < 0.01）。肌生成素和 MHC 的表达降低了约 50%。尽管存在 EpoR mRNA 和蛋白，但 rhEpo 也不能逆转低氧对 L6 细胞或人肌母细胞的影响。
数据表明，暴露于低氧条件（1% O2）下的大鼠和人肌母细胞改变了它们的增殖和分化过程。它们还表明，Epo 不是一种有效的生长因子来对抗这种有害影响。
