Trolox对猪肌肉干细胞增殖及分化的影响

Effects of Trolox on Proliferation and Differentiation of Pig Muscle Stem Cells

【目的】探究抗氧化剂-水溶性维生素E的类似物(Trolox)通过调控活性氧对猪肌肉干细胞增殖及分化过程产生的影响,为进一步优化培养肉种子细胞在体外增殖及分化过程提供研究基础。【方法】首先在猪肌肉干细胞增殖过程中,添加不同浓度(0、50、100和200μmol·L^(-1))的Trolox培养3 d,利用血细胞计数板进行细胞计数,统计细胞增殖倍数,同时利用CCK8技术检测不同浓度的Trolox对细胞增殖的影响;利用RT-qPCR技术检测不同浓度Trolox处理后表征干性的PAX7表达水平,Western Blotting技术检测PAX7蛋白的表达水平;通过CellROX荧光染料对细胞内活性氧进行染色并通过高通量高内涵活细胞共聚焦成像系统检测Trolox对活性氧的调控作用;进一步在猪肌肉干细胞体外成肌分化进程中添加Trolox处理,利用RT-qPCR技术检测分化早期标志基因MYOG、CAV-3及终末分化标志基因肌球蛋白重链(MyHC)的表达水平,并利用Western Blotting技术检测MyHC蛋白的表达,利用免疫荧光技术对MyHC进行染色并统计MyHC阳性细胞比例。【结果】细胞增殖倍数统计显示,50和100μmol·L^(-1)的Trolox处理组猪肌肉干细胞增殖倍数显著高于对照组(P<0.05);CCK8测得50和100μmol·L^(-1) Trolox处理组第3天的吸光值显著高于对照组(P<0.05);100和200μmol·L^(-1)的Trolox显著上调了PAX7的表达(P<0.05),对PAX7蛋白的表达有上调趋势,但无显著差异(P>0.05);添加Trolox后,细胞内活性氧水平被极显著降低(P<0.001);在分化进程中添加Trolox后,预分化阶段的MYOG和CAV-3及终末分化阶段的MyHC均显著上调(P<0.05),而Western blotting结果显示MyHC蛋白的表达无显著变化(P>0.05);免疫荧光结果显示MyHC阳性细胞比例有增多的趋势,但无显著差异(P>0.05)。【结论】Trolox通过降低细胞内的活性氧,促进猪肌肉干细胞的增殖以及分化进程。

【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 200μmol·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 100μmol·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 100μmol·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 200μmol·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.