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A kind of method of c2c12 myoblast differentiation culture

A technology of myoblasts and differentiation medium, which is applied in the field of C2C12 myoblasts differentiation culture, can solve the problems of long-term passage preservation and utilization of cells, and the reduction of myoblast proliferation and differentiation ability, so as to achieve good differentiation effect and improve utilization rate effect

Inactive Publication Date: 2017-12-26
河南护理职业学院
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Problems solved by technology

However, the myoblasts isolated by this method are primary myoblasts. After the mouse skeletal myoblasts were isolated and passaged for more than 5 times, they were induced to differentiate in 2% horse serum for three days, and only a small amount of myoblasts could be formed. Tube
Therefore, after the primary myoblasts separated by the existing method have been passaged for 4-5 times, the myoblasts will lose the ability to form myotubes in vitro, and the proliferation and differentiation capabilities of the myoblasts will be severely reduced. There is no way for cells to be preserved for long-term passage

Method used

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  • A kind of method of c2c12 myoblast differentiation culture

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[0027] The preparation method of arctiin is as follows: the medicinal material of arctium is crushed to 40-50 mesh, add 10 times of water, reflux and extract twice, each time for 2 hours; combine the filtrate, filter the filtrate, and then apply D101 resin for adsorption and elution, and the elution is sequential Elute with 2 times the resin volume of water, 20% ethanol and 50% ethanol, collect the 50% ethanol eluate, then concentrate to remove ethanol, add 0.5% activated carbon after concentration and boil for 30min, centrifuge to remove activated carbon, static at 0-4°C Set overnight, filter and dry, that is.

[0028] The preparation method of Rehmannia glutinosa polysaccharide is as follows: Rehmannia glutinosa is crushed to 40-50 mesh particles, petroleum ether is degreased, monosaccharides and oligosaccharides are removed with 80% ethanol, the dregs are heated to evaporate the solvent, and then according to the ratio of solid to liquid 1:60, Cellulase 3%, adjust the pH to...

Embodiment 1

[0032] A method for culturing C2C12 myoblasts, comprising the following steps:

[0033] (1) According to the passage method above, take continuous passage C2C12 myoblasts, inoculate them into culture flasks containing growth medium, and place them in CO 2 cultured in an incubator with 95% air and 5% CO 2 , the temperature was 37°C, the medium was changed every 48 hours, and the cell morphology was observed using an inverted microscope;

[0034] (2) When the cells grow to the logarithmic phase, discard the old medium, wash the cells twice with PBS, then add 1mL of 0.25% trypsin-EDTA to cover the cells, and the digested cells can be observed under an inverted microscope. If the cells are about to separate and appear round, suck out the trypsin to stop the digestion, pat the culture bottle lightly to make the cells fall off the wall of the bottle, add 3m L of growth medium, blow gently with a pipette several times to break up the cell clumps, Aspirate the cell suspension, put i...

Embodiment 2

[0040] It is basically the same as in Example 1, except that mouse myoblasts of 6 consecutive passages are used, and the growth medium is a high-sugar DMEM medium as the base fluid, containing 10% fetal bovine serum, 1% double antibody, Arctiin 9μg / L, EGF 0.2mg / L, Insulin 100mg / L, Rehmannia polysaccharide 50mg / L.

[0041] The differentiation medium was based on high-sugar DMEM medium, containing 2% horse serum, 1% double antibody, 8 μg / L daidzein, 20 mg / L pomegranate extract, and 10 mg / L honeysuckle extract.

[0042] Mouse myoblasts of passage 6 were inoculated in growth medium and observed under a microscope. Two days later, spindle-shaped, translucent cells with good refraction were visible under the microscope. The cells grew to 80% confluence. Small cells inoculated in differentiation medium In mouse myoblasts, small myotubes formed after a day and a half, such as figure 1 As shown, more than 85% of the cultured cells differentiated into mature myotubes after five days. ...

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Abstract

The invention belongs to differentiation culture of myoblasts, and relates to a differentiation culture method of C2C12 myoblasts. Separated mouse myoblasts sub-cultured more than five times are used as objects, a growth medium and a differentiation medium are optimized through steps of cell proliferation culture, differentiation culture and the like, and over 85% of the myoblasts sub-cultured more than five times can differentiate myotubes, thus effectively improving the utilization ratio of the separated primary myoblasts.

Description

technical field [0001] The invention belongs to the differentiation and cultivation of myoblasts, and relates to a method for the differentiation and cultivation of C2C12 myoblasts. Background technique [0002] C2C12 is a mouse myoblast, which differentiates rapidly and can form contractile myotubes and produce characteristic myosin. Therefore, it is widely used in various medical research, such as biomedicine, clinical research on muscle regeneration, etc. The study of myoblast differentiation is important for understanding muscle cell development and repair (regeneration). Myoblasts in culture can exhibit features of the myogenesis process, including proliferation, migration, fusion, myotube formation, and contraction. Muscle cells grown in medium containing 10% fetal bovine serum continued to proliferate even when confluent; however, when they were grown in medium containing 2-5% horse serum, they fused and formed myotubes. The mouse cell line C2C12 is a cell line wid...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/077
CPCC12N5/0658C12N2500/30C12N2500/34C12N2500/76C12N2501/11C12N2501/33C12N2501/90C12N2501/999
Inventor 何敏陈旭王艾青黄晓璐付平姬新颖李民友高东霞
Owner 河南护理职业学院
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