山羊胎儿肌肉干细胞的分离培养与成肌诱导分化

doi:10.3864/j.issn.0578-1752.2018.08.016

Abstract

Abstract: 【Objective】 To establish the method for isolation, culture, identification and myogenic differentiation of fetal muscle stem cells from Anhuai goat in vitro, and to provide experimental materials for further research on the molecular mechanism of goat muscle stem cell proliferation and differentiation. 【Method】 In this study, the goat fetal longissimus muscle tissue was selected and cut into meat emulsion with ophthalmology, digested with 0.1% type I collagenase for 40 min and then digested with 0.25% trypsin for 15 min. The isolated cells were cultured in growth medium (20% FBS + 80% DMEM / F12 + Penicillin) in a 37℃, 5% CO2 incubator. After culturing for 2h, the cells were purified by differential adherent technique. After 2h, the cells were purified again. The cells were subcultured when they reached 70% density. 30min adherent method was used for further purification with each subculture of muscle stem cells until the sixth passages. Muscle stem cell marker genes Pax7 and MyoD1 were detected with the purified cells for identification. When muscle stem cells grew to a density of about 70%, the growth medium was displaced with differentiation medium (2% FBS + 98% DMEM / F12 + Penicillin)for myoblasts induction and the morphology of the cells was monitored . One day after the induction of the cells, the marker protein of muscle stem cells Myog was detected. In addition, total RNAs of cells induced at 0, 1, 3, 5, and 7 days were separately extracted and their relative expression amounts of MyoD1 and Myog genes were measured by qPCR. 【Result】 Isolated cells showed adherent growth, and their morphology tended to be long spindle after stabilization. Pax7 and MyoD1 expression were detected in the 6th passage cells by immunofluorescence. After induction by differentiated medium, the cells started to differentiate and fuse with each other into myotubes with a certain directionality as the induction prolonged. Myog protein was detected by immunofluorescence assay. Differentiation marker genes MyoD1 and Myog were detected by qPCR. Expression of MyoD1 could be detected in the first day of induction and maintained until the 3rd day, and its level began to decline from the 5th day but still significantly higher than the proliferative phase. A similar dynamic was observed with the relative expression level of Myog in the differentiating cells.【Conclusion】In this experiment, the fetal muscle stem cells of Anhuai goat was obtained with high purity, which showed good myogenic potential after induction. The results provide material for further research on the mechanisms of myogenic differentiation of muscle stem cells.

Key words: goat fetus, muscle stem cells, isolation culture, identification, myogenic differentiation

SUI MengHua, ZHENG Qi, WU Hao, DING JianPing, LIU Yong, LI WenYong, CHU MingXing, ZHANG ZiJun, LING YingHui. Isolation, Culture and Myogenic Differentiation of Muscle Stem Cells in Goat Fetal[J].Scientia Agricultura Sinica, 2018, 51(8): 1590-1597.

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Isolation, Culture and Myogenic Differentiation of Muscle Stem Cells in Goat Fetal

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