Abstract
Aim: This study aimed to determine the effect of mechanical ventilation (MV) on the differentiation and proliferation of diaphragm satellite cells. Methods: Diaphragm satellite cells were isolated from C57 mice receiving 6 h of MV with optimized magnetic-activated cell sorting (MACS) approach. The cells were stained with BrdU or antibody for differentiation marker MYH3. The expression of MyoD and myogenin was detected by real-time PCR. Results: Diaphragm satellite cells were successfully isolated from mice by using MACS with a set of optimized parameters. About 1.5 × 105 cells could be harvested from a diaphragm. Upon MV, the proliferation rate of diaphragm satellite cells was decreased from 88.74% to 81.92%, while the differentiation rate was increased from 17.94% to 27.58%, compared to controls. Moreover, the expression of MyoD and myogenin were significantly upregulated upon MV. Conclusions: We established a practical method to purify diaphragm satellite cells, and demonstrated that MV regulated the differentiation and proliferation of diaphragm satellite cells.
Highlights
Previous study reported that pro-myogenic factors or the transplantation of muscle satellite cells (MuSCs) can enhance skeletal muscle regeneration [14]
4.1 MV inhibited the proliferation of diaphragm satellite cells
Using MACS, 1.5 × 105 diaphragm satellite cells could be isolated from a diaphragm using the optimized parameters
Summary
Previous study reported that pro-myogenic factors or the transplantation of muscle satellite cells (MuSCs) can enhance skeletal muscle regeneration [14]. Several protocols have been published to isolate MuSCs from skeletal muscle [15, 16]. The diaphragm is distinct from other skeletal muscles because it is activated constantly to drive ventilation and diaphragm satellite cells are myogenic cells with potential for proliferation and differentiation [17]. It is difficult to isolate diaphragm satellite cells because of the challenge to preserve the quiescence state of diaphragm satellite cells during the isolaton. In this study we established an optimized method to purify diaphragm satellite cells, and determined the effects of MV on the differentiation and proliferation of satellite cells
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