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首页> 外文期刊>Cell and Tissue Research >IGF-I binding and receptor signal transduction in primary cell culture of muscle cells of gilthead sea bream: changes throughout in vitro development
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IGF-I binding and receptor signal transduction in primary cell culture of muscle cells of gilthead sea bream: changes throughout in vitro development

机译：银头鲷鱼肌肉细胞原代细胞培养中的IGF-I结合和受体信号转导：整个体外发育过程中的变化

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摘要

We examined the possibility of culturing muscle cells of gilthead sea bream in vitro and assessed variations in insulin-like growth factor-I (IGF-I) binding during myocyte development. The viability of the cell culture was determined by fluorescence-activated cell-sorting analysis, which showed that the percentage of dead cells decreased with cell differentiation. The intracellular reduction of MTT into formazan pigment was preferentially carried out as cells differentiated (from day 4) indicating an increase in metabolic activity. IGF-I-binding assays demonstrated that the number of receptors increased from 190 ± 0.09 fmol/mg protein in myocytes at day 5 to 360 ± 0.09 fmol/mg protein in myotubes at day12. The affinity of IGF-I receptors did not change significantly during cell development (from 0.89 ± 0.09 to 0.98 ± 0.09 nM). The activation of various kinase (ERK 1/2 MAPK and Akt/PKB) proteins by IGFs and insulin was studied by means of Western blot analysis. Levels of MAPK-P increased after IGF and insulin treatment during the first stages of cell culture, with a low response being observed at day 15, whereas IGFs displayed a stimulatory effect on Akt-P throughout the cell culture period, even on day 15. This study thus shows that (1) gilthead sea bream myocytes can be cultured, (2) they express functional IGF-I receptors that increase in number as they differentiate in vitro; (3) IGF signalling transduction through IGF-I receptors stimulates the MAPK and Akt pathways, depending on the development stage of the muscle cell culture.
机译：我们检查了在体外培养金头鲷的肌肉细胞的可能性，并评估了心肌细胞发育过程中胰岛素样生长因子-I（IGF-I）结合的变化。通过荧光激活的细胞分选分析确定细胞培养物的活力，这表明死亡细胞的百分比随着细胞分化而降低。当分化的细胞（从第4天开始）表明代谢活性增加时，优先进行MTT向甲maz色素的细胞内还原。 IGF-I结合试验表明，受体数量从第5天的肌细胞中的190±0.09 fmol / mg蛋白增加到第12天的肌管中的360±0.09 fmol / mg蛋白。在细胞发育过程中，IGF-I受体的亲和力没有明显变化（从0.89±0.09到0.98±0.09 nM）。通过蛋白质印迹分析研究了胰岛素样生长因子和胰岛素对各种激酶（ERK 1/2 MAPK和Akt / PKB）蛋白的激活。在细胞培养的第一阶段，在IGF和胰岛素治疗后，MAPK-P的水平增加，在第15天观察到低响应，而IGF在整个细胞培养期间甚至在第15天都表现出对Akt-P的刺激作用。因此，这项研究表明（1）可以培养金头鲷的心肌细胞；（2）它们表达的功能性IGF-I受体在体外分化时数量会增加； （3）取决于肌肉细胞培养物的发育阶段，通过IGF-I受体的IGF信号转导刺激了MAPK和Akt途径。

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