高密度培养对猪肌肉干细胞命运的影响

Effect of High-density Culture on the Fate of Porcine Muscle Stem Cells

以猪肌肉干细胞为研究对象,通过接种4组不同初始密度(2.7×10^(3)、5.4×10^(3)、1.1×10^(4)cells/cm2和2.2×10^(4)cells/cm^(2))细胞培养3 d,探究高密度条件对猪肌肉干细胞命运的影响,其中2.7×10^(3)cells/cm^(2)组为对照组。计数结果表明,猪肌肉干细胞适宜的培养密度为2×10^(4)~3×10^(4)cells/cm^(2),超出此范围造成了增殖相关蛋白p-ERK1/2的下调,从而产生增殖抑制效应;转录组测序结果显示最高密度组与对照组相比共有2 125个差异表达基因,主要富集在细胞衰老、细胞周期和PI3K-Akt等信号通路;深入研究转录组学结果发现,高密度条件下干性基因PAX7、PAX3和静息相关基因SPRY1表达上调;分化基因MYOD、MYOG、MYH3和衰老基因P21、P53、P15出现上调表达。实时聚合酶链式反应和Western Blot结果显示,随着接种密度升高,干性基因PAX7和SPRY1、分化基因MYOG、衰老基因P21均出现上调表达。以上结果表明,猪肌肉干细胞在高密度条件下,会出现重返静息状态、分化和衰老等多种细胞命运。本研究结果有助于理解高密度下细胞命运决定机制,为细胞培养肉研究在高密度条件下调节细胞增殖分化提供一定的理论基础。

In this study,four different initial densities(2.7×10^(3) as a control,5.4×10^(3),1.1×10^(4),and 2.2×10^(4) cells/cm^(2))of porcine muscle stem cells were cultured for three days to explore the effect of high-density culture conditions on the fate of porcine muscle stem cells.The counting results showed that the suitable culture density of porcine muscle stem cells was 2×10^(4)–3×10^(4) cells/cm^(2),and the proliferation-related protein phosphorylated extracellular signal-regulated kinase 1/2(p-ERK1/2)was down-regulated when the initial cell density was outside this range,causing a proliferation inhibitory effect.The transcriptome sequencing results showed that 2125 differentially expressed genes were identified between the highest density and control groups,mainly enriched in some signaling pathways such as those related to cell senescence and cell cycle and the PI3K-Akt signaling pathway.Furthermore,the expression of the stemness genes PAX7 and PAX3 and the quiescence-related gene SPRY1 was upregulated under high-density conditions.The expression of the differentiation genes MYOD,MYOG and MYH3 and the senescence genes P21,P53 and P15 was upregulated.The results of real-time PCR and Western blot assay showed that the expression of PAX7,SPRY1,MYOG,and P21 were upregulated as the cell density increased.The above results showed that porcine muscle stem cells had multiple fats such as returning to a quiescent state,differentiation and senescence under high-density conditions.The results of this study can help in understanding the mechanism of cell fate determination and provide a theoretical basis for studying the regulation of cell proliferation and differentiation under high-density condition.