CN114317417B - Method for inducing skeletal muscle satellite cells to generate lipid droplets - Google Patents

Abstract

The invention belongs to the technical field of cells, and particularly relates to a method for inducing skeletal muscle satellite cells to generate lipid droplets. The method comprises the following steps: skeletal muscle satellite cells are collected from the dorsum muscles of piglets which are just born for 1-5d, purified, and lipogenesis induced when the purified skeletal muscle satellite cells are attached to and converged to 60% -70%. The method can effectively induce the porcine skeletal muscle satellite cells to generate lipid droplets. By adopting the method provided by the invention, the pig skeletal muscle satellite cells have obvious lipid production effect, so that the pork quality can be effectively improved.

Description

Method for Inducing Skeletal Muscle Satellite Cells to Produce Lipid Droplets

technical field

The invention belongs to the field of cell technology, and in particular relates to a method for inducing skeletal muscle satellite cells to generate lipid droplets.

Background technique

Meat production is an important part of animal husbandry. With the continuous improvement of living standards, the demand for meat products is increasing day by day. How to improve the level of meat production has become an important task for scientific and technological workers ("Skeletal muscle secretion affects pig muscle growth and metabolism Regulation", Chen Jie, Compilation of Abstracts of the Tenth National Symposium on Animal Physiology and Biochemistry, 2008, page 10, line 1-2 of the abstract, open date June 10, 2009).

However, the meat quality of existing pigs is not good.

Contents of the invention

In view of this, the purpose of the present invention is to provide a method for inducing skeletal muscle satellite cells to generate lipid droplets.

To achieve the above object, the technical solution of the present invention is:

A method for inducing skeletal muscle satellite cells to generate lipid droplets, comprising the following steps:

Skeletal muscle satellite cells were collected from the dorsal muscle of newborn 1-5d piglets, and purified. When the purified skeletal muscle satellite cells reached 60%-70% confluence, adipogenic induction was performed.

Further, the purification adopts a differential attachment method.

Further, the lipogenesis induction comprises the following steps:

On the 1st-2d, add dexamethasone, insulin, 3-isobutyl-1-methylxanthine and double antibody to the basal medium, and culture for 2d;

On the 3rd-4th day, add insulin, rosiglitazone and double antibodies to the basal medium, and culture for 2 days;

On the 5th-6th day, add dexamethasone, insulin, 3-isobutyl-1-methylxanthine and double antibody to the basal medium, and culture for 2 days;

On the 7th-8th day, add insulin, rosiglitazone and double antibodies to the basal medium, and culture for 2 days;

On the 9th-10th day, glucose and double antibodies were added to the basal medium, and cultured for 2 days.

Further, the basal medium adopts DMEM/F12 medium.

Further, on day 1-2, add 0.5-1 μmol/L dexamethasone, 5-10 μg/mL insulin, 0.1-0.5 mmol/L 3-isobutyl-1-methylxanthine and 1% -1.5% double antibody, cultivated for 2 days, the percentage is volume percentage.

Further, on the 3rd to 4th day, add 5-10 μg/mL insulin, 5-10 uM rosiglitazone and 1%-1.5% double antibody to the basal medium, culture for 2 days, and the percentages are volume percentages.

Further, on the 5th-6th day, add 0.5-1 μmol/L dexamethasone, 5-10 μg/mL insulin, 0.1-0.5 mmol/L 3-isobutyl-1-methylxanthine and 1% -1.5% double antibody, cultivated for 2 days, the percentage is volume percentage.

Further, on the 7th-8th day, 5-10 μg/mL insulin, 5-10 uM rosiglitazone and 1%-1.5% double antibody were added to the basal medium, and cultured for 2 days, the percentages are volume percentages.

Further, on the 9th-10th day, add 5-10 mM glucose and 1%-1.5% double antibody to the basal medium, and culture for 2 days, and the percentages are volume percentages.

The beneficial effects of the present invention are:

The method of the invention can effectively induce pig skeletal muscle satellite cells to generate lipid droplets.

By adopting the method of the invention, the satellite cells of the porcine skeletal muscle have an obvious lipogenic effect, thereby effectively improving the pork quality.

The method of the invention is simple and convenient to operate.

Description of drawings

Fig. 1 is the morphology of the pure skeletal muscle satellite cell that embodiment 1 obtains;

Fig. 2 is the PAX-7 fluorescence staining figure and cell nucleus staining figure of embodiment 2, wherein 2A and 2B are fluorescence staining figure (*10) and cell nucleus staining figure (*10) respectively;

Fig. 3 is the oil red O staining result figure of embodiment 4; Wherein, 3A and 3B are the oil red O staining result figure of 2d (*10) and 2d (*20) respectively;

Fig. 4 is the oil red O staining result figure of embodiment 4; Wherein, 4A and 4B are the oil red O staining result figure of 4d (*10) and 4d (*20) respectively;

Fig. 5 is the oil red O staining result figure of embodiment 4; Wherein, 5A and 5B are the oil red O staining result figure of 6d (*10) and 6d (*20) respectively;

Fig. 6 is the oil red O staining result figure of embodiment 4; Wherein, 6A and 6B are the oil red O staining result figure of 8d (*10) and 8d (*20) respectively;

Fig. 7 is the oil red O staining result figure of embodiment 4; Wherein, 7A and 7B are the oil red O staining result figure of 10d (*10) and 10d (*20) respectively.

Detailed ways

The examples given are for better description of the content of the present invention, but the content of the present invention is not limited to the examples given. Therefore, non-essential improvements and adjustments to the implementation by those skilled in the art based on the content of the invention above still fall within the scope of protection of the present invention.

The following DMEM/F12 medium, fetal bovine serum FBS, L-Glutamax and Non-Essentia Amino AcidsSolution are from Gibco, USA;

The following basic fibroblast growth factor bFGF comes from Shanghai Biyuntian Biotechnology Co., Ltd.;

The following chicken embryo extract CEE comes from Gemini Company in the United States;

The following dexamethasone, IBMX, insulin, rosiglitazone, double antibody, polylysine, oil red O, goat anti-mouse Pax7, fluorescent mouse secondary antibody and glucose were from Solarbio.

Example 1

The specific steps for obtaining pig skeletal muscle satellite cells are as follows:

Slaughter the newborn piglets, quickly take the half-bond muscles and back muscles of each individual, and immediately put them in Hanks solution, pack them and do the next step;

Cut out the required skeletal muscle in the clean bench, wash with PBS containing 1% double antibody, and remove connective tissues such as fat, muscle bonds, and sarcolemma as much as possible;

Cut into pieces to about 2 mm3 and transfer to a centrifuge tube, blow and beat with PBS 3 times, settle naturally for 5 minutes, and discard the supernatant; add 0.2% collagenase, digest in a 37°C water bath for 50 minutes, shake once every 5 minutes, and wash with PBS three times after the end. Centrifuge at 1000r/min for 10min, discard the supernatant; then digest with an appropriate amount of 0.25% trypsin in a 37% water bath for 20min, shake once every 5min, and immediately add an equal volume of medium (DMEM/F12+20 %FBS + 1% double antibody) to stop the digestion, filter through 100-mesh, 200-mesh and 400-mesh cell sieves successively, centrifuge the filtered suspension at 1000r/min for 10min, discard the supernatant, and use the precipitated cells for growth Culture medium is inoculated in cell culture dish; Gained cell suspension is added in culture dish after digestion, at 37 ℃, 5% CO _2Cultivate in cell culture box (used every 100ml medium composition is: 80%DMEM/F12, 20 %FBS, 0.5% CEE, 1% GlutaMax, 1% NEAA, 1% double antibody, 2.5ul bFGF) after 2h, then slowly suck out the culture solution and add it to a new pre-coated poly-lysine Petri dish, 12h Afterwards, the culture solution was sucked out, and then added to a new culture bottle to obtain pure skeletal muscle satellite cells, the morphology of which is shown in Figure 1.

It can be seen from Figure 1 that skeletal muscle satellite cells are long spindle-shaped.

Example 2

After the pure skeletal muscle satellite cells obtained in Example 1 were fixed in 4wt% paraformaldehyde cell fixative for 15 minutes, the cells were permeabilized with 0.25% TritonX-100, followed by rinsing with PBS for 3 times, and then adding 3wt% BSA Block for 1 hour, add primary antibody PAX-7, incubate overnight at 4°C, absorb primary antibody PAX-7, wash with PBST three times, add fluorescent secondary antibody, incubate for one hour in the dark, then observe under an inverted fluorescence microscope, as shown Figure 2 shows.

It can be seen from Figure 2 that PAX-7 was positively expressed in green fluorescence, and the nuclei were in blue. This proves that the cells obtained in Example 1 are indeed skeletal muscle satellite cells.

Example 3

To induce adipogenesis of porcine skeletal muscle satellite cells, the specific steps are as follows:

A. Collect skeletal muscle satellite cells from the dorsal muscle of piglets just born 1-5 days old, and purify the cells through differential adhesion. After the purified skeletal muscle satellite cells are adhered to 60%-70% of the confluence, they will be induced by lipogenesis ;

B. On the 1-2d day of induction of differentiation, add 1 μmol/L dexamethasone, 5 μg/mL insulin, 0.5 mmol/L 3-isobutyl-1-methylxanthine and 1% to DMEM/F12 basal medium Double antibody, cultivated for 2 days;

C. On the 3rd-4th day of induction of differentiation, add 5 μg/mL insulin, 10uM rosiglitazone and 1% double antibody to the DMEM/F12 basal medium, and culture for 2 days;

D. On day 5-6 of induction of differentiation, add 1 μmol/L dexamethasone, 5 μg/mL insulin, 0.5 mmol/L 3-isobutyl-1-methylxanthine and 1% to DMEM/F12 basal medium Double antibody, cultivated for 2 days;

E. On the 7th-8th day of induction of differentiation, add 5 μg/mL insulin, 10 uM rosiglitazone and 1% double antibody to the DMEM/F12 basal medium, and culture for 2 days;

F. On the 9th-10th day of induction of differentiation, add 10 mM glucose and 1% double antibody to the DMEM/F12 basal medium, and culture for 2 days;

The stated percentages all refer to volume percentages.

Example 4

The lipid droplets produced by the cells of Example 3 were subjected to an oil red staining test according to the instruction manual of the oil red O staining kit, and the results are shown in Figures 3-6; among them, Figure 3 is the result of Oil Red O staining in 2d; Figure 4 is the result of 4d Oil red O staining results; Figure 5 is the 6d oil red O staining results; Figure 6 is the 8d oil red O staining results; Figure 7 is the 10d oil red O staining results.

It can be seen from Figure 3-7 that the pig skeletal muscle satellite cells have an obvious lipogenic effect.

In addition, it should be understood that although this specification is described according to implementation modes, not each implementation mode only includes an independent technical solution, and this description in the specification is only for clarity, and those skilled in the art should take the specification as a whole , the technical solutions in the various embodiments can also be properly combined to form other implementations that can be understood by those skilled in the art.

Claims (7)

1. A method for inducing skeletal muscle satellite cells to produce lipid droplets, comprising the steps of: collecting skeletal muscle satellite cells from the dorsum muscles of piglets which are just born for 1-5d, purifying, and performing adipogenesis induction when the purified skeletal muscle satellite cells are converged to 60% -70% by adherence; the purification adopts a differential adherence method; the lipogenesis inducing method comprises the following steps:

1 st to 2 nd, adding dexamethasone, insulin, 3-isobutyl-1-methylxanthine and diabody into the basal medium, and culturing for 2d;

3 rd to 4 th days, adding insulin, rosiglitazone and double antibody into the basic culture medium, and culturing for 2d;

5 th to 6 th days, adding dexamethasone, insulin, 3-isobutyl-1-methylxanthine and diabody into the basal medium, and culturing for 2d;

7 th to 8 th days, adding insulin, rosiglitazone and double antibody into the basic culture medium, and culturing for 2d; and 9-10d, adding glucose and double antibodies into the basal medium, and culturing for 2d.

2. The method of claim 1, wherein the basal medium is DMEM/F12 medium.

3. The method according to claim 1 or 2, characterized in that 0.5-1 μmol/L dexamethasone, 5-10 μg/mL insulin, 0.1-0.5 mmol/L3-isobutyl-1-methylxanthine and 1% -1.5% diabody are added to the basal medium for 2d, said percentages being volume percentages.

4. A method according to claim 3, characterized in that 5-10 μg/mL insulin, 5-10uM rosiglitazone and 1% -1.5% diabody are added to the basal medium for 3-4d, incubated for 2d, said percentages being volume percentages.

5. The method according to claim 4, wherein 0.5-1. Mu. Mol/L dexamethasone, 5-10. Mu.g/mL insulin, 0.1-0.5 mmol/L3-isobutyl-1-methylxanthine and 1% -1.5% diabody are added to the basal medium for 2d, said percentages being by volume.

6. The method of claim 5, wherein the basal medium is supplemented with 5-10 μg/mL insulin, 5-10uM rosiglitazone and 1% -1.5% diabody for 2d at 7-8d, the percentages being by volume.

7. The method of claim 6, wherein 5-10mM glucose and 1% -1.5% diabody are added to the basal medium for 9-10d, and the basal medium is cultured for 2d, and the percentages are volume percentages.

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