Ciliary neurotrophic factor combined with forskolin and 3-isobutyl-1-methylxanthine induced muscle derived stem cells to differentiate into Schwann cells phenotype through cyclic adenosine monophosphate signaling pathway

doi:10.12307/2022.373

Abstract

Abstract: BACKGROUND: Schwann cells are the most ideal seed cells for peripheral nerve tissue engineering, but Schwann cells have many defects, so the research of cell therapy to repair peripheral nerve injury mainly focuses on other cells with Schwann cells characteristics, and Schwann cells-like cells have become the main source of choice for peripheral nerve injury repair.
OBJECTIVE: To investigate the method of differentiation of muscle derived stem cells into Schwann cells phenotypes induced by ciliary neurotrophic factor combined with forskolin and 3-isobutyl-1-methylxanthine and the correlation between the induction and the cyclic adenosine monophosphate signaling pathway, so as to lay the foundation for further application of muscle derived stem cells in peripheral nerve injury repair.
METHODS: Muscle derived stem cells were extracted from one-week old SD rats with mixed digestive enzymes. Dedifferentiation of muscle derived stem cells was induced by ciliary neurotrophic factor, and then the dedifferentiated muscle derived stem cells were induced by 3-isobutyl-1-methylxanthine. Cell morphology, immunofluorescence, CCK-8 assay, western blot assay, RT-qPCR and other techniques were used to identify and analyze muscle derived stem cells, dedifferentiated cells and Schwann cells-like cells. The expression of proteins related to cyclic adenosine monophosphate signaling pathway during dedifferentiation and Schwann cells-like differentiation was detected.
RESULTS AND CONCLUSION: (1) Cell immunofluorescence staining showed that primary cells taken from SD rats could be labeled with Desmin, a specific marker of muscle derived stem cells. CCK-8 assay results showed that the proliferation activity of P1-3 muscle derived stem cells gradually increased, and the proliferation activity of P3-8 muscle derived stem cells tended to stabilize. (3) Western blot assay showed that the expression of myogenic differentiation-related proteins p21 and MyoG decreased after ciliary neurotrophic factor induction, and the expression of cyclic adenosine monophosphate-related protein p-CREB increased. After 3-isobutyl-1-methylxanthine induction, the expression levels of Schwann cells markers S100β, GFAP, p75 and p-CREB increased, while cyclic adenosine monophosphate inhibitor SQ22536 could inhibit the effects of ciliary neurotrophic factor and 3-isobutyl-1-methylxanthine. (4) The RT-qPCR results were consistent with the western blot results; that was, the expression levels of S100β, GFAP and p75 mRNA increased after 3-isobutyl-1-methylxanthine induction, and SQ22536 could inhibit this effect. The results of CCK-8 assay showed that the cell activity was reduced after 3-isobutyl-1-methylxanthine induction. (5) Results concluded that ciliary neurotrophic factor combined with 3-isobutyl-1-methylxanthine can induce muscle derived stem cells to differentiate into Schwann cells phenotype, and the induction process activates the cyclic adenosine monophosphate signaling pathway.

Key words: stem cells, muscle derived stem cells, ciliary neurotrophic factor, forskolin, 3-isobutyl-1-methylxanthine, Schwann cells, cyclic adenosine monophosphate, signaling pathway

CLC Number:

Gong Chao, Zhi Xiaodong, Zhang Yuqiang, Wang Chenliang, Wang Kang, Wang Wei. Ciliary neurotrophic factor combined with forskolin and 3-isobutyl-1-methylxanthine induced muscle derived stem cells to differentiate into Schwann cells phenotype through cyclic adenosine monophosphate signaling pathway[J]. Chinese Journal of Tissue Engineering Research, 2022, 26(19): 2970-2977.

Figures/Tables 8

2.6 FSK-IBMX诱导去分化肌源性干细胞分化为许旺细胞样细胞 Fsk-IBMX诱导过程中可见诱导组和抑制组细胞随着培养时间的延长，细胞数量均逐渐减少，而对照组细胞则相反，细胞密度随着培养时间的延长明显增加，见图6A-N。Fsk-IBMX诱导后24 h，诱导组和抑制组细胞折光性增强，呈神经元样改变，细胞间有突触样结构形成，见图6E和6F，而对照组有少数细胞折光性增强，细胞形态无明显变化，见图6D；诱导后72 h，诱导组和抑制组细胞折光性明显增强，形态变化更为明显，细胞聚集成神经球，诱导组较抑制组更为聚集，突起明显增长并交织成网，见图6H-J，而对照组细胞折光性较24 h减弱，有轻微聚集现象，但无神经球形成，细胞生长有一定的方向性，见图6G；诱导后120 h，诱导组和抑制组神经球细胞折光性更强，更加聚集，交织成网的突起明显增粗，但抑制组均弱于诱导组，见图6L-N，而对照组细胞折光性较72 h无明显变化，部分细胞有轻微聚集现象，细胞变得细长，生长有明显的方向性，见图6K。 Fsk-IBMX诱导去分化肌源性干细胞120 h后，Western blot结果显示，与对照组相比，诱导组细胞S100β、GFAP和p75均明显增加(P < 0.05)，而抑制组S100β、GFAP和p75的表达较对照组无明显变化，与诱导组相比均明显降低(P < 0.01，P < 0.05和P < 0.05)，见图6O，P。RT-qPCR结果与Western blot结果一致，即诱导组细胞S100β、GFAP和p75 mRNA表达水平较对照组均明显增加(P < 0.001，P < 0.001和P < 0.000 1)，而抑制组与诱导组相比mRNA表达水平均明显降低(P < 0.01，P < 0.001和P < 0.000 1)，见图6Q。结果表明，Fsk-IBMX可以诱导去分化肌源性干细胞分化为许旺细胞样细胞，而SQ22536可以抑制这种诱导作用。 CCK-8法检测显示，与对照组相比，FSK-IBMX抑制了去分化肌源性干细胞的细胞活性(P < 0.000 1)，而加入SQ22536后，这种抑制作用并未被明显减弱，见图6R。为了证实睫状神经营养因子和Fsk-IBMX相继处理后，肌源性干细胞确实已发生了神经分化，对肌源性干细胞特异性标志物Desmin的表达水平进行了Western blot检测，结果显示，经睫状神经营养因子复合Fsk-IBMX诱导后，Desmin的表达水平显著降低(P < 0.01)，见图6S和6T。"

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