关键词: Trolox, 猪肌肉干细胞, 活性氧, 增殖, 分化

Abstract:

【Objective】The objective of this study was to investigate the regulatory effects of Trolox, an antioxidant water-soluble vitamin E analogue, on the proliferation and differentiation of pig muscle stem cells, which possibly was affected by reactive oxygen species. The study could provide a theoretical fundament for further optimizing the proliferation and differentiation process of cultured meat seed cells in vitro. 【Method】Initially, 0, 50, 100 and 200 μmol·L^-1 of Trolox was added to pig muscle stem cells during their proliferation culture for 3 d, respectively. The blood cell counter and CCK8 technology were both used to detect the influence of Trolox on the cell proliferation. RT-qPCR was employed to measure the expression level of PAX7 gene for the further characterizing cellular stemness induced by Trolox. Meanwhile, Western Blotting was also used to testify the expression level of PAX7 protein. The intracellular reactive oxygen species was stained by CellROX fluorescent dye, and the High-throughput High-content Live Cell Confocal Imaging System was adopted to evidence the regulatory effect of Trolox on reactive oxygen species. Trolox was additionally used to the in vitro myogenic differentiation of pig muscle stem cells. RT-qPCR was used to detect the expression levels of early differentiation marker genes MYOG, CAV-3 and terminal differentiation marker gene myosin heavy chain (MyHC). Western Blotting was applied to detect the expression of MyHC protein, whereas Immunofluorescence technique stained MyHC and counted the proportion of MyHC positive cells. 【Result】The cell proliferation fold statistics indicated that the proliferation fold of pig muscle stem cells in 50 or 100 μmol·L^-1 Trolox treatment groups was significantly higher than that under the control group (P<0.05); CCK8 test showed that the absorbance value under 50 or 100 μmol·L^-1 Trolox treatment groups on the 3rd day was significantly higher than that under the controls (P<0.05); Trolox concentrations at 100 or 200 μmol·L^-1 significantly up-regulated the expression of PAX7 gene (P<0.05), but had no dominant effect for the improvement of PAX7 proteins with no statistical difference (P>0.05). The level of reactive oxygen species in the cells was significantly reduced (P<0.001) when Trolox applied. Moreover, after the addition of Trolox to the cells in their differentiation process, MYOG and CAV-3 in the predifferentiation stage as well as MyHC genes in the terminal differentiation stage were dramatically up-regulated (P<0.05). However, Western Blotting results exhibited that the expression of MyHC protein had no a huge change (P>0.05), while the immunofluorescence results displayed that the proportion of MyHC positive cells had an increasing trend but there was no statistical difference (P>0.05). 【Conclusion】Trolox promoted the proliferation and differentiation of pig muscle stem cells via reducing reactive oxygen species in the cells.

Key words: Trolox, pig muscle stem cells, reactive oxygen species, proliferation, differentiation