鹅骨骼肌卫星细胞的分离培养与鉴定

doi:10.11843/j.issn.0366-6964.2023.10.017

Abstract

Abstract: The purpose of the study was to establish methods for isolation, culture, purification, and identification of goose skeletal muscle satellite cells in vitro. The healthy 10-week-old Xupu geese was selected as experimental materials. Satellite cells were isolated from the muscle with the action of Dispase II and collagenase II. Satellite cells were purified by differential adhesion to the culture dish. During in vitro culture, the complete DMEM/F12 growth medium containing 20% FBS and supplemented with bFGF could better maintain the differentiation potential of satellite cells, and the culture dishes involved in the culture process needed to be coated with matrigel specially. After the formation of mature myotubes, the expression of myosin heavy chain (MyHC) was detected by immunofluorescence and Western blotting. The relative expression levels of Pax7, MyoD1 and MyoG before (growth medium, GM) and after differentiation (differentiation medium, DM) were detected by qRT-PCR. There were 3 replicates in each group before and after differentiation. The results showed that the newly isolated satellite cells were small in size and strong in refraction. After adhering to the wall, they had a spindle-shaped structure with needle-like ends and puffed up in the middle. The cells were identified as Pax7 positive by immunofluorescence. Mature multinucleated myotubes could be formed after induction of differentiation. MyHC was positive by immunofluorescence detection. The statistical differentiation index was as high as 78% (P<0.01). The marker genes Pax7, MyoD1, and MyoG were detected positive by qRT-PCR. The relative expression of Pax7 gene before differentiation was 2.22 times (P<0.01) that after differentiation, while the relative expression of marker genes MyoD1 and MyoG after differentiation were 1.90 (P<0.01) and 44.22 times (P<0.01) that before differentiation, respectively. Western blotting results showed that the expression level of MyHC protein was significantly increased after differentiation. In this study, a method for the isolation and culture of goose skeletal muscle satellite cells by collagenase II and dispase II was established. The culture system can enable cells to maintain huge differentiation potential during the in vitro culture. The study results provide a good cell model for the research of the growth and development molecular regulation mechanism of goose muscle tissue.

Key words: goose, skeletal muscle satellite cells, isolation, culture, identification

CLC Number:

S835

ZHANG Li, XU Jialong, HUANG Jinyu, XU Ziyue, LEI Xinnuo, LU Huipeng, ZHU Rui, SUN Weixiang, CAO Haiyue, WANG Anping, ZHU Shanyuan. Isolation, Culture, and Identification of Skeletal Muscle Satellite Cells in Goose[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(10): 4186-4195.

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Isolation, Culture, and Identification of Skeletal Muscle Satellite Cells in Goose

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