Scientia Agricultura Sinica ›› 2020, Vol. 53 ›› Issue (8): 1664-1676.doi: 10.3864/j.issn.0578-1752.2020.08.015

• ANIMAL SCIENCE·VETERINARY SCIENCE·RESOURCE INSECT • Previous Articles     Next Articles

Isolation, Culture, Identification and Biological Characteristics of Pig Skeletal Muscle Satellite Cells

QIN BenYuan,YANG Yang,ZHANG YanWei,LIU Min,ZHANG WanFeng,WANG HaiZhen,WU YiQi,ZHANG XueLian,CAI ChunBo,GAO PengFei,GUO XiaoHong,LI BuGao,CAO GuoQing()   

  1. College of Animal Science and Veterinary Medicine, Shanxi Agricultural University, Taigu 030801, Shanxi
  • Received:2019-09-29 Accepted:2020-01-13 Online:2020-04-16 Published:2020-04-29
  • Contact: GuoQing CAO E-mail:anniecao710502@aliyun.com

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Abstract:

【Objective】 The aim of this study was to establish a method for isolation, purification and identification of porcine skeletal muscle satellite cells in vitro, and to explore its biological characteristics, in order to provide a reliable cell model for further research of muscle growth and development in pigs. 【Method】 In this study, the longissimus dorsi muscle of 1 day old pig was selected and cut into meat emulsion in aseptic state. After that, it was digested with 0.2% type I collagenase for 90 min, and then digested with 0.25% trypsin for 30 min at 37 ℃. After termination of digestion, filtration and resuspension, the isolated cells were cultured in a 37 ℃ and 5% CO2 cell incubator. The skeletal muscle satellite cells were purified by repeated differential adherence technique. The first purification selection was performed after cell culturing for 2 h, and non-adherent cells were transferred to a new culture dish. After the supernatant was further cultured for 18 h, the satellite cells were purified again. The cells were subcultured or frozen when the cell density reached 70% - 80%. Cell immunofluorescence technique was used to detect the protein expression of marker genes of Pax7 and MyoD of P2 satellite cell, and the growth curve was determined. The satellite cells were differentiated into myocytes, adipocytes and osteoblasts by adding different inducing differentiation fluids. The protein expression of myoblast differentiation marker gene MHC was detected by using cell immunofluorescence to identify myotube formation in satellite cells. Oil red O staining and triglyceride content were quantified to identify the adipogenic differentiation effect in satellite cells. Alizarin red staining was used to identify the osteogenic differentiation ability in satellite cells, which were detected the expression of key genes during myogenesis, adipogenesis and osteogenesis by qRT-PCR. 【Result】 The results showed that the satellite cells with higher purity were isolated and purified by two-step enzymatic digestion and repeated differential adherence technique. The cells that were originally isolated with highly refractive, and were fusiform or spindle-shaped after adherence, after which the cells extended and began to proliferate. The results of cell immunofluorescence identification of satellite cells specific marker proteins Pax7 and MyoD were positive, indicating that the isolated cells were skeletal muscle satellite cells. Skeletal muscle satellite cell proliferation underwent the incubation period, and the growth period and finally reached the plateau phase. The satellite cells were self-fusion when the cells grew to 90% density. After myogenic induction and differentiation of the satellite cells, a large number of myotubes were formed by the adjacent satellite cells. Multinucleated myotubes were regularly arranged and the myoblast marker protein MHC staining was positive. The qRT-PCR results showed that the marker genes of MyoD and MyoG both increased first and then decreased during the process of myoblast differentiation. After adipogenic induction, the cell morphology changed into triangle, and lipid droplets appeared and aggregated into large lipid droplets with continuous induction. Oil red O staining observed a large number of red grape-like lipid droplets. Oil red O staining quantitative results showed that the triglyceride content steadily increasing during the adipogensis. There were extremely significant differences among each time point (P<0.01). The qRT-PCR results showed that the expression level of PPARγ gene was high in the middle and the late stage of induction. The FABP4 gene reached the highest at the 6th day of induction and was significantly higher than that at other time points (P<0.01). A similar dynamic was observed with the relative expression level of CEBP/β and HSL in the differentiating cells. Their expression tended to increase first and then decrease. After inducing osteogenic differentiation, it was found that the cell morphology became irregular. Cells formed bone nodules after inducing, compared with the period without induction, the alizarin red staining showed that the number and density of round opaque calcified nodules were significantly increased. The results showed that the cells appeared osteogenic differentiation. The expression levels of osteogenic marker genes BGLAP and RUNX2 also showed a steady upward trend during the inducing procession, which was significantly different from that without inducing cells (P<0.01). 【Conclusion】 This study established a method for isolation and purification of pig skeletal muscle satellite cells based on combined enzyme digestion and differential adherent technique. The obtained cells had strong proliferation ability and multi-directional differentiation potential. The results provided a technical platform for pig skeletal muscle satellite cells as seed cells for future tissue engineering research.

Key words: pig, satellite cell, isolation culture, identification, differentiation

Table 1

Primers for qRT-PCR"

基因名称Gene name 引物序列（5′→3′）Primer sequence 产物长度Product length (bp) 序列号Accession No.
MyoD F: GAGAGCACTACAGCGGTGAC
R: CCTCGCTGTAATAGGTGCCG		 132 NM_001002824.1
MyoG F: CTCAGCTCCCTCAACCAGGA
R: GGAGTGCAGATTGTGGGCAT		 195 NM_001012406.1
FABP4 F:TGGTACAGGTGCAGAAGTGG
R: TTCTGGTAGCCGTGACACCT		 108 NM_001002817.1
CEBP/β F:CTGGAGACGCAGCATAAGGT
R:TGCTTGAACAAGTTCCGCAG		 110 NM_001199889.1
PPARγ F:CTATTCCATGCTGTCATGGGTG
R: ACCATGGTCACCTCTTGTGA		 107 NM_214379.1
HSL F:CACTGACTGCTGACCCCAAG
R:TCCTCACTGTCCTGTCCTTCAC		 121 NM_214315.3
BGLAP F: GCCACACTCTGCCTTGCTG
R: CTCCTGGAAGCCGATGTGA		 237 NM_001164004.1
RUNX2 F: GAGTGGACGAGGCAAGAGTT
R:ACTTGGTGCAGAGTTCAGGG		 256 XM_003482203.4
18S F:CCCACGGAATCGAGAAAGAG
R: TTGACGGAAGGGCACCA		 122 NW_018085108.1

Fig. 1

The morphology of skeletal muscle satellite cells (100×) A: The separated skeletal muscle satellite cells are spherical and have strong refractive index; B: Some satellite cells begin to adhere and extend to the periphery; C: 48 h of satellite cells are fusiform or spindle-shaped; D: Separated and cultured for 72 h. The number of satellite cells increased and converged; E: Morphology of cells growth to 70%; F:Cells elongated and thinned regularly after cultured 5 days; G: 7 days of satellite cells spontaneously fused to form multinucleated myotubes; H: Recovery skeletal muscle satellite cells frozen after 3 passages"

Fig. 2

Immunofluorescence identification of skeletal muscle satellite cells (100×) A: Pax7 expression was positive; B: DAPI stained nuclei; C: Image merged between A and B; D: MyoD expression was positive; E: DAPI stained nuclei; F: Image merged between D and E"

Fig. 3

Skeletal muscle satellite cell growth curve"

Fig. 4

Myogenic differentiation of skeletal muscle satellite cells and immunofluorescence (100×) A: After 48 h induction, satellite cells fusion and a amount of myotubes were appeared; B: Induced 72 h, the number of myotubes increased; C: After 4 days induction, myotubes were fused together; D: MHC immunofluorescence; E: DAPI stained nuclei; F: Image merged between D and E"

Fig. 5

Expression of MyoD and MyoG after myogenic differentiation Different uppercase letters indicate that extremely significant differences (P<0.01). The same as below"

Fig. 6

Adipogenic differentiation of skeletal muscle satellite cells and Oil red O staining A-D: Morphological observation of satellite cells at 3, 6, 9, 12 d induced by adipogenesis (100×); E-H: The lipid droplets was positive expression under Red Oil O dyed (100×); I-L: After the induction, the red precipitate was observed at the bottom of the culture dish"

Fig. 7

Oil red O staining quantitative results"

Fig. 8

Expression of PPARγ, CEBP/β, FABP4 and HSL after adipogenic differentiation"

Fig. 9

Osteogenic differentiation of skeletal muscle satellite cells and Alizarin Red staining A-C: After osteogenic induction 7, 14, 21 d, satellite cells were observed under microscope (100×); D-F: Alizarin red staining can stain the calcified nodules formed after induction into red (100×); G-I: Macroscopic observation of red precipitated nodules at the bottom after the osteogenesis"

Fig. 10

Expression of BGLAP and RUNX2 after osteogenic induction"

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