Microfluidic analysis of extracellular matrix-bFGF crosstalk on primary human myoblast chemoproliferation, chemokinesis, and chemotaxis.
Microfluidic analysis of extracellular matrix-bFGF crosstalk on primary human myoblast chemoproliferation, chemokinesis, and chemotaxis.
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DOI：
10.1039/c5ib00060b
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发表时间：
2015-05
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影响因子：
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通讯作者：
Heilshorn SC
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作者：
Ferreira MM;Dewi RE;Heilshorn SC
成肌细胞暴露于碱性成纤维细胞生长因子(碱性成纤维细胞生长因子)，这是肌肉损伤后释放的结果，导致受体磷酸化，更快的迁移，并促进增殖。这些影响发生在跨越三个数量级(100-103分钟)的时间尺度上。传统上用于评估趋化性的Transwell分析的有限元模型显示，跨膜孔形成的bFGF梯度是短暂的，在第一分钟内减少了45%。因此，为了评估碱性成纤维细胞生长因子诱导的超过102分钟的迁移，我们使用了一种能够产生稳定的线性浓度梯度的微流控分析来执行单细胞趋化和趋化分析。我们假设潜在的细胞外基质(ECM)的组成可能影响成肌细胞对可溶性bFGF的行为反应，因为以前对其他细胞类型的研究表明整合素和成纤维细胞生长因子(FGF)受体之间存在交叉作用。与这一概念一致，我们发现bFGF显著减少了成肌细胞在层粘连蛋白上培养的倍增时间，但不能减少纤维连接蛋白或胶原蛋白的倍增时间。层粘连蛋白的迁移速度(13.4μm/h)明显快于纤维连接蛋白(10.6μm/h)和胶原蛋白(7.6μm/h)。碱性成纤维细胞生长因子驱动的化学运动以严格相加的方式进一步加快了迁移速度，导致所有受试ECM的平均增加2.3μm/h。我们观察到相对轻微的化学吸引(约67%的成肌细胞)对3.2 ng/mL/mm的碱性成纤维细胞生长因子梯度的反应，而不考虑ECM的特性。因此，虽然细胞外基质-碱性成纤维细胞生长因子的串扰确实影响了趋化细胞的增殖，但对趋化作用或趋化作用没有明显的影响。这些数据表明，碱性成纤维细胞生长因子对成肌细胞迁移的主要生理作用是化学运动，衰老和/或疾病引起的周围ECM的变化可能通过改变成肌细胞的迁移和增殖来影响肌肉再生。
Exposing myoblasts to basic fibroblast growth factor (bFGF), which is released after muscle injury, results in receptor phosphorylation, faster migration, and increased proliferation. These effects occur on time scales that extend across three orders of magnitude (100 – 103 minutes). Finite element modeling of Transwell assays, which are traditionally used to assess chemotaxis, revealed that the bFGF gradient formed across the membrane pore is short-lived and diminishes 45% within the first minute. Thus, to evaluate bFGF-induced migration over 102 minutes, we employed a microfluidic assay capable of producing a stable, linear concentration gradient to perform single-cell analyses of chemokinesis and chemotaxis. We hypothesized that the composition of the underlying extracellular matrix (ECM) may affect the behavioral response of myoblasts to soluble bFGF, as previous work with other cell types has suggested crosstalk between integrin and fibroblast growth factor (FGF) receptors. Consistent with this notion, we found that bFGF significantly reduced the doubling time of myoblasts cultured on laminin but not fibronectin or collagen. Laminin also promoted significantly faster migration speeds (13.4 μm/h) than either fibronectin (10.6 μm/h) or collagen (7.6 μm/h) without bFGF stimulation. Chemokinesis driven by bFGF further increased migration speed in a strictly additive manner, resulting in an average increase of 2.3 μm/h across all ECMs tested. We observed relatively mild chemoattraction (~ 67% of myoblast population) in response to bFGF gradients of 3.2 ng/mL/mm regardless of ECM identity. Thus, while ECM-bFGF crosstalk did impact chemoproliferation, it did not have a significant effect on chemokinesis or chemotaxis. These data suggest that the main physiological effect of bFGF on myoblast migration is chemokinesis and that changes in the surrounding ECM, resulting from aging and/or disease may impact muscle regeneration by altering myoblast migration and proliferation.