In-vitro culture method and identification method of micropterus japonicus skeletal muscle satellite cells

A technology of satellite cells and in vitro culture, applied in cell dissociation methods, bone/connective tissue cells, animal cells, etc., can solve problems such as research gaps

Pending Publication Date: 2021-12-24
OCEAN UNIV OF CHINA
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The purpose of the embodiments of the present invention is to provide an in vitro culture method and identification method of the perch skeletal muscle satellite cells, aiming to solve the problem that there is a blank research on the cultivation of the perch skeletal muscle satellite cells in the prior art

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  • In-vitro culture method and identification method of micropterus japonicus skeletal muscle satellite cells
  • In-vitro culture method and identification method of micropterus japonicus skeletal muscle satellite cells
  • In-vitro culture method and identification method of micropterus japonicus skeletal muscle satellite cells

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Embodiment 1

[0056] In the present embodiment, the specific steps of culturing skeletal muscle satellite cells of perch are as follows:

[0057] 1) Primary culture of cells: ① Immerse the individual perch used for sampling in 75% ethanol for 30s, then take a small piece of muscle above the lateral line of the perch and below the dorsal fin, and transfer it into a medium containing 4% third antibody (BI, no. PB180424) in a 50ml sterile centrifuge tube of PBS (Biosharp, BL601A). Transfer the tissue pieces to a sterile 50ml centrifuge tube containing 75% ethanol and soak for 30s, then pass through 50ml centrifuge tubes containing 4%, 3%, 2%, and 1% third-antibody PBS in turn, soak for 5min each time, and shake the centrifuge tube during , so that the tissue block and PBS are fully contacted and sterilized; ② Move the tissue block to a container containing 1% third-antibody PBS; cut the tissue block to about 1mm 3 ; ③ Leave the tissue block solution after step ② to be shredded, discard the up...

Embodiment 2

[0063] Example 2 Identification method of sea bass skeletal muscle satellite cell types and stages

[0064] The method for identification of the cell types cultured in Example 1. In this example, muscle satellite cells before induction of differentiation and cells fused into myotubes after induction of differentiation were collected. Trizol method (Vazyme, R401-01) was used to extract the total number of cells in two stages. RNA, through the reverse transcription kit (Vazyme, P611-01), 1ug ​​cellular RNA was reverse-transcribed into cDNA, and the marker gene pax7 of skeletal muscle satellite cells, the marker genes myod1 and myod2 of the proliferation stage and the marker genes of the differentiation stage were amplified. The marker gene myogenin is used to determine the cell type and stage. The PCR system is 5uL Mix (Vazyme, P213-01), pax7a, pax7b, myod1, myod2, myogenin upstream and downstream primers 0.4uL each, 1uL cDNA, add water to make up to 10uL, and then perform PCR A...

Embodiment 3

[0068] Example 3 Identification method for the differentiation ability of sea bass skeletal muscle satellite cells

[0069] The method for identifying the differentiation ability of passaged cells obtained: After the cells induced to differentiate for 72 hours were washed 1-2 times with PBS, fixed with 4% paraformaldehyde for 30 minutes, washed with PBS for 5 minutes × 3 times, washed with 0.25% Triton X-100 ( Solarbio, T8200) was permeabilized with PBS for 20 min, washed with PBS for 5 min x 3 times, added 0.5 mg / ml DAPI (Solarbio, C0065) for staining for 2 min, and observed under a fluorescent inverted microscope. It can be seen that multinucleated muscle cells appear, indicating that the cultured muscle satellite cells have good ability to induce differentiation in vitro ( Figure 5 , wherein, A is the light microscope image of the perch muscle satellite cells induced and differentiated for 72 hours, and B is the DAPI staining image of the perch muscle satellite cells induc...

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Abstract

The invention is applicable to the technical field of biology, and provides an in-vitro culture method and an identification method of micropterus japonicus skeletal muscle satellite cells. The micropterus japonicus skeletal muscle satellite cells are successfully subjected to in-vitro separation culture, the purity is higher after differential adherence, subculture can be performed, besides, the micropterus japonicus skeletal muscle satellite cells can be effectively induced, differentiated and fused into multi-core muscle tubes, functional muscle fibers are finally developed, and experimental materials can be provided for molecular mechanisms of growth and development of seawater sclerochloas muscle, functional verification of related genes and research on skeletal muscle injury repair.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an in vitro culture method and an identification method of perch skeletal muscle satellite cells. Background technique [0002] Lateolabrax maculatus, commonly known as perch, green perch, village flower, seven-star perch, etc., belongs to Perciformes (Perciformes), Lipidaceae (Serranidae), and Lateolabrax genus (Lateolabrax). High, widely loved by consumers. In fish, skeletal muscle accounts for about 40%-60% of the weight of the whole body, and the growth of skeletal muscle plays a decisive role in the growth of fish body. Skeletal muscle originates from somites developed from paraaxial mesoderm during embryonic development, and its basic structural unit is muscle fiber. The formation of skeletal muscle mainly undergoes the muscle development process in the embryonic period and the muscle growth process in the post-embryonic period. From the perspective of muscle fibe...

Claims

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Application Information

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IPC IPC(8): C12N5/077C12Q1/02
CPCC12N5/0659G01N33/5005C12N2509/00
Inventor 齐鑫李昀温海深张静茹董夕梦张凯强
Owner OCEAN UNIV OF CHINA
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