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Isolated culture method of horse skeletal muscle satellite cells

A satellite cell, separation and culture technology, applied in the direction of cell dissociation method, bone/connective tissue cells, tissue culture, etc., can solve the problem of lack of separation and culture method of equine skeletal muscle satellite cells, and achieve the effect of great scientific research and social value

Inactive Publication Date: 2020-04-14
INNER MONGOLIA AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] For this reason, the embodiment of the present invention provides a method for isolating and culturing equine skeletal muscle satellite cells to solve the problem of lack of an effective method for isolating and culturing equine skeletal muscle satellite cells in the prior art

Method used

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  • Isolated culture method of horse skeletal muscle satellite cells
  • Isolated culture method of horse skeletal muscle satellite cells
  • Isolated culture method of horse skeletal muscle satellite cells

Examples

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Effect test

Embodiment 1

[0036] Example 1, Isolation and Culture of Horse Skeletal Muscle Satellite Cells

[0037] The method for isolating and culturing horse skeletal muscle satellite cells of the embodiment of the present invention comprises the following steps:

[0038] 1. Collect the muscle of a 2-year-old horse, soak and sterilize the horse muscle quickly with 70% ethanol in a petri dish, transfer the sterilized muscle sample to a new petri dish containing DPBS containing penicillin and streptomycin immediately, and use Wash with 3 times the volume of DPBS until the DPBS is clear and bloodless. Among them, DPBS should be added with 1% penicillin and streptomycin in advance and filtered through a 0.22 μm filter.

[0039] 2. Remove the visible fat and connective tissue on the washed horse muscle mass with a scalpel, chop the blocky horse muscle tissue with scissors in a petri dish containing DMEM, and cut the muscle tissue to 1mm 3 size.

[0040] The shredded muscle tissue was transferred to a ...

Embodiment 2

[0045] Example 2. Identification of horse skeletal muscle satellite cells by induced differentiation method

[0046] When the equine skeletal muscle satellite cells cultured in Example 1 of the present invention reached 70%-80% confluency, differentiation culture medium with low serum was added to induce differentiation. Such as figure 2 As shown in the results, it was found that after the differentiation medium was used to induce differentiation, the cultured cells were regularly arranged in parallel, the cells elongated and became thinner, fusion occurred between a large number of proliferating cells, and the formation of myotubes could be observed, which was consistent with Differentiation characteristics of skeletal muscle satellite cells.

Embodiment 3

[0047] Example 3. Identification of equine skeletal muscle satellite cells by differential attachment method

[0048] In the process of embodiment 1 cell culture may be subject to the contamination of fibroblasts, the equine skeletal muscle satellite cells are purified by the differential attachment method, the attachment time of equine skeletal muscle satellite cells is longer, and the attachment time of fibroblasts is very short. Fast, observation of attachment time of skeletal muscle satellite cells and fibroblasts, and identification of equine skeletal muscle satellite cells based on this characteristic. Such as image 3 As shown in the results, it was found that no cells of horse skeletal muscle satellite cells attached to the wall at 1 hour, but a small number of fibroblasts began to attach to the wall at 1 hour, and horse skeletal muscle satellite cells just started to attach to the wall at 4 hours, forming Fibroblasts have begun to adhere to the wall, and the cell sha...

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Abstract

The embodiment of the invention discloses an isolated culture method of horse skeletal muscle satellite cells. The isolated culture method comprises the following steps: sterilizing horse muscle blocks, removing adipose tissues and connective tissues, and crushing into blocky muscle tissues; digesting the blocky muscle tissue by trypsin, and performing centrifugal separation to obtain a first supernatant and a first precipitate; and centrifuging the first supernatant to obtain a second supernatant and a second precipitate, discarding the second supernatant, re-suspending and centrifuging the second precipitate with a proliferation culture solution to obtain a third supernatant and a third precipitate, discarding the third supernatant, suspending the proliferation culture solution of the third precipitate, filtering through a cell filter, collecting a first filtrate, and centrifuging the first filtrate to obtain a fourth precipitate. According to the embodiment of the invention, the quantity and the purity of the skeletal muscle satellite cells are improved through purification, and an accurate skeletal muscle satellite cell isolated culture method is provided for scientific research colleges and universities for research of muscle growth, muscle injury and the like.

Description

technical field [0001] The embodiment of the present invention relates to the technical field of cell culture, and in particular to a culture system of horse skeletal muscle satellite cells. Background technique [0002] At present, horse racing and recreational horses are the mainstream of the development of the horse industry in the future, and the muscles of horses will be damaged to varying degrees during exercise according to the amount of exercise, which will affect the performance of horses. In horse racing and equestrian sports, horses have Good athletic performance is a must. Skeletal muscle satellite cells are myogenic stem cells with proliferation and differentiation in skeletal muscle, which play an indispensable role in the regeneration of skeletal muscle. Most of the skeletal muscle satellite cells are quiescent after adulthood, and will only grow in tissue When damaged, it will be activated to proliferate, differentiate, and promote the repair of muscle fiber...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/077
CPCC12N5/0659C12N2509/00
Inventor 格日乐其木格赵雅丽张心壮才文道力玛曹迪纳日嘎芒来
Owner INNER MONGOLIA AGRICULTURAL UNIVERSITY
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