Tissue Engineering Centre, Faculty of Medicine, Universiti Kebangsaan Malaysia, Jalan Yaacob Latif, Cheras, 56000, Kuala Lumpur, Malaysia.
Department of Physiology, Faculty of Medicine, Universiti Kebangsaan Malaysia, Jalan Yaacob Latif, Cheras, 56000, Kuala Lumpur, Malaysia.
Tissue Eng Regen Med. 2020 Dec;17(6):835-845. doi: 10.1007/s13770-020-00283-3. Epub 2020 Aug 6.
One of the long-standing problems of myoblasts in vitro expansion is slow cell migration and this causes fibroblast population to exceed myoblasts. In this study, we investigated the synergistic effect of laminin and epidermal growth factor (EGF) on co-cultured myoblasts and fibroblasts for cell attachment, proliferation and migration.
Skeletal human muscle cells were cultured in four different conditions; control, EGF, laminin (Lam) and laminin EGF (Lam + EGF). Using live imaging system, their cellular properties; attachment, migration and growth were exposed to Rho kinase inhibitor, Y-27632, and EGF-receptor (EGF-R) inhibitor, gefitinib were measured.
Myoblast migration and proliferation was enhanced significantly by synergistic stimulation of laminin and EGF (0.61 ± 0.14 µm/min, 0.008 ± 0.001 h) compare to that by EGF alone (0.26 ± 0.13 µm/min, 0.004 ± 0.0009 h). However, no changes in proliferation and migration were observed for fibroblasts among the culture conditions. Inhibition of Rho kinase resulted in the increase of the myoblast migration on the laminin-coated surface with EGF condition (0.64 ± 0.18 µm/min). Compared to the untreated conditions, myoblasts cultured on the laminin-coated surface and EGF demonstrated elongated morphology, and average cell length increase significantly. In contrast, inhibition of EGF-R resulted in the decrease of myoblast migration on the laminin coated surface with EGF supplemented condition (0.43 ± 0.05 µm/min) in comparison to the untreated control (0.53 ± 0.05 µm/min).
Laminin and EGF preferentially enhance the proliferation and migration of myoblasts, and Rho kinase and EGF-R play a role in this synergistic effect. These results will be beneficial for the propagation of skeletal muscle cells for clinical applications.
体外扩增的成肌细胞存在一个长期存在的问题，即细胞迁移缓慢，这导致成纤维细胞数量超过成肌细胞。在这项研究中，我们研究了层粘连蛋白和表皮生长因子（EGF）对共培养的成肌细胞和成纤维细胞的协同作用，以观察细胞黏附、增殖和迁移。
在四种不同条件下培养骨骼肌细胞：对照、EGF、层粘连蛋白（Lam）和层粘连蛋白+表皮生长因子（Lam+EGF）。使用活细胞成像系统，观察 Rho 激酶抑制剂 Y-27632 和表皮生长因子受体（EGF-R）抑制剂吉非替尼对细胞附着、迁移和生长的影响。
与单独使用 EGF 相比，层粘连蛋白和 EGF 的协同刺激显著增强了成肌细胞的迁移和增殖（0.61±0.14μm/min，0.008±0.001h）。然而，在这些培养条件下，成纤维细胞的增殖和迁移没有变化。在存在 EGF 的情况下，抑制 Rho 激酶导致在层粘连蛋白涂覆表面上的成肌细胞迁移增加（0.64±0.18μm/min）。与未经处理的条件相比，在层粘连蛋白涂覆表面上培养的成肌细胞呈拉长形态，平均细胞长度显著增加。相比之下，在存在 EGF 的情况下，EGF-R 抑制剂的存在导致补充 EGF 的层粘连蛋白涂覆表面上的成肌细胞迁移减少（0.43±0.05μm/min），与未经处理的对照相比（0.53±0.05μm/min）。
层粘连蛋白和 EGF 优先增强成肌细胞的增殖和迁移，Rho 激酶和 EGF-R 在这种协同作用中发挥作用。这些结果将有益于骨骼肌细胞的临床应用。
