CN112094806A - Method for improving adherent culture growth speed of equine skeletal muscle satellite cells and cell oxidative stress culture model - Google Patents
Method for improving adherent culture growth speed of equine skeletal muscle satellite cells and cell oxidative stress culture model Download PDFInfo
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Abstract
The invention discloses a method for improving the adherent culture growth speed of equine skeletal muscle satellite cells, which comprises the steps of adding a gelatin solution into a culture container, and adding 5% CO at 37 DEG C2Placing for 1-3h in the environment to obtain a culture container with gelatin layer formed on the surface; transferring the equine skeletal muscle satellite cells to the culture container with gelatin layer formed on the surface, adding proliferation culture solution, 37 ℃ and 5% CO2Culturing in the environment for 1-3h to obtain adherent culture equine skeletal muscle satellite cells. The method of the invention accelerates the adherent speed of the skeletal muscle satellite cells, shortens the adherent time of the skeletal muscle satellite cells and accelerates the acquisition of the skeletal muscle satellite cells. The cell oxidation model provides favorable conditions and research foundation for researching muscle growth, muscle oxidation injury and other scientific research institutions, provides skeletal muscle satellite cells for repairing muscle injury, and has great scientific research and social values.
Description
Technical Field
The invention relates to the technical field of cell culture, in particular to a method for improving the adherent culture growth speed of equine skeletal muscle satellite cells and a cell oxidative stress culture model.
Background
With the change of the social development and production mode in China, the modern horse industry mainly develops the race horse, equestrian sports and riding entertainment, the state and all levels of governments have also paid attention to the development of the modern horse industry, the horse industry is definitely an important component of the animal husbandry, and the horse competitive sports industry is actively developed. A large amount of oxygen free radicals generated in the process of horse movement can cause oxidative stress, so that the structure and permeability of a muscle cell membrane are changed, muscle damage is caused, and the movement performance of the horse is reduced. The skeletal muscle satellite cells are myogenic stem cells which have proliferation and differentiation in skeletal muscle and play an essential role in the skeletal muscle regeneration process, but on one hand, the skeletal muscle satellite cells have the limitations or disadvantages of long culture time, difficult acquisition and the like in the culture process. On the other hand, the cell oxidative stress model has important significance for researching the principle of oxidative stress of livestock and the in vitro research of oxidative damage.
At present, no horse skeletal muscle oxidative stress model is published. Therefore, it is highly desirable to develop a cell model and a cell oxidative stress model that are important for studying muscle growth and development, injury and repair, diseases associated with oxidative stress, and the like.
Disclosure of Invention
Therefore, the invention provides a method for improving the adherent culture growth speed of equine skeletal muscle satellite cells and a cell oxidative stress culture model.
In order to achieve the above purpose, the invention provides the following technical scheme:
a method for improving the growth speed of adherent culture of equine skeletal muscle satellite cells comprises the steps of adding a gelatin solution into a culture vessel, and adding 5% CO at 37 DEG C2Placing for 1-3h in the environment to obtain a culture container with gelatin layer formed on the surface;
transfer of the equine skeletal muscle satellite cells to the surface to formAdding proliferation culture solution into culture container with gelatin layer, and culturing at 37 deg.C and 5% CO2Culturing in the environment for 1-3h to obtain adherent culture equine skeletal muscle satellite cells.
In one embodiment of the present invention, after removing the excess gelatin solution from the culture container having the gelatin layer formed on the surface thereof, the culture container is washed twice with DPBS, and the proliferation culture solution is added thereto.
In one embodiment of the invention, the mass concentration of the gelatin solution is 0.0025 g/ml.
In one embodiment of the invention, the proliferation solution is 20% fetal bovine serum, 1% penicillin streptomycin and amphotericin B, DMEM supplemented.
The invention also provides an equine skeletal muscle satellite cell oxidative stress culture model, and H is added into the adherent culture equine skeletal muscle satellite cell prepared by the method2O2The horse skeletal muscle satellite cell oxidative stress culture model is obtained by culturing in the stimulation culture solution.
In one embodiment of the present invention, the hydrogen-containing compound contains H2O2In the stimulated culture solution of (1), H2O2Concentrate 600. mu. mol/L.
In one embodiment of the invention, the adherent culture equine skeletal muscle satellite cells are cultured in the culture vessel with the gelatin layer formed on the surface to reach 70% -80% confluence.
In one embodiment of the invention, the addition comprises H2O2The time for culturing in the stimulated culture solution of (2) to (10) hours.
The invention has the following advantages:
the embodiment of the invention provides a method for improving the adherent culture growth speed of equine skeletal muscle satellite cells and a cell oxidative stress culture model, obtains equine skeletal muscle satellite cells which are subjected to rapid adherent proliferation and have a good growth state, and establishes the cell oxidative stress model by using the equine skeletal muscle satellite cells which are subjected to adherent proliferation and have a good growth state.
The method of the invention accelerates the adherent speed of skeletal muscle satellite cells, shortens the adherent time of the skeletal muscle satellite cells, and accelerates the acquisition of skeletal muscle satellite cellsAnd (4) cells. The invention utilizes H2O2The cells are induced to generate oxidative stress to establish a cell oxidation model, a rapid establishment method of the skeletal muscle satellite cell oxidation model is provided for research institutions such as muscle growth and muscle oxidative damage, beneficial conditions and research bases are provided for the skeletal muscle satellite cell to be used for muscle damage repair, and the method has great scientific and social values.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It should be apparent that the drawings in the following description are merely exemplary, and that other embodiments can be derived from the drawings provided by those of ordinary skill in the art without inventive effort.
Fig. 1 is a microscopic image of the anchorage velocity of skeletal muscle satellite cells with and without gelatin layering according to an embodiment of the present invention, in which a: adhering the gelatin-free skeletal muscle satellite cells for 1 h; b: gelatin-free skeletal muscle satellite cells for 4 h; c: gelatin-free skeletal muscle satellite cells for 8 h; d: laying gelatin for skeletal muscle satellite cell adherence for 1 h; e: laying gelatin for skeletal muscle satellite cell adherence for 4 h; f: paving gelatin for skeletal muscle satellite cell adherence for 8 h;
FIG. 2 is a diagram of H according to an embodiment of the present invention2O2Graph of the effect of treatment on ROS in skeletal muscle satellite cells, with red line at 0. mu. mol/L for 6 hours of treatment, green line at 800. mu. mol/L for 4 hours, blue line at 600. mu. mol/L for 6 hours, and yellow line at 800. mu. mol/L for 6 hours.
Detailed Description
The present invention is described in terms of particular embodiments, other advantages and features of the invention will become apparent to those skilled in the art from the following disclosure, and it is to be understood that the described embodiments are merely exemplary of the invention and that it is not intended to limit the invention to the particular embodiments disclosed. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In the embodiment of the invention, the preparation method of the gelatin solution is 0.0025g/ml, the gelatin solution is prepared by deionized water and sterilized by an autoclave, the gelatin needs to be spread in a culture dish one hour in advance, and the gelatin solution can be used after being washed twice by DPBS before use.
In the embodiment of the invention, the preparation method of the proliferation culture solution comprises the following steps: 20% Fetal Bovine Serum (FBS), 1% penicillin streptomycin and amphotericin B, DMEM supplemented and filtered through 0.22 μm filters.
In the examples of the present invention, H is contained2O2The preparation method of the culture solution comprises the following steps: mixing 30% of H2O20.5ml of the solution was diluted to 4.5ml of deionized water and mixed to prepare 1mol/L (1000 mmol/L) of H2O2Stock solution is prepared into H with different concentrations2O2Stock solutions (0, 100, 200, 400, 600, 800 and 1000 mmol/L). Different concentrations of H2O2Adding 15 μ l of stock solution into 14.85ml of culture solution for proliferation of skeletal muscle satellite cells to obtain H2O2The action concentrations are respectively 0, 100, 200, 400, 600, 800 and 1000 mu mol/L, and the mixture is filtered by a 0.22 mu m filter after being uniformly mixed.
In the examples of the present invention, DMEM was purchased from Gibco, Inc. under the trade designation 2066482. DPBS was purchased from Gibco, Inc. under the trade name 2043115, and was prepared as a DPBS solution in the form of a powder in bags, each bag of DPBS being dissolved in 1L of distilled water.
The apparatus used in the examples of the present invention is as follows: TD6A-WS desk centrifuge, HWS-26 model electric heating constant temperature water bath, Thermo Scientific HERAcell 150i CO2Incubator, ZEISS inverted phase contrast microscope, NovoCyte flow cytometer.
In the embodiment of the invention, the mass concentration of the gelatin solution is 0.0025g/ml, the gelatin solution is prepared by deionized water and sterilized by an autoclave, the gelatin needs to be spread in a culture dish one to three hours in advance, and the gelatin can be used after being washed twice by DPBS before use.
Example 1 optimized culture of equine skeletal muscle satellite cells
The method for improving the growth speed of the equine skeletal muscle satellite cell adherent culture provided by the embodiment of the invention comprises the following steps:
step one, culturing an equine skeletal muscle satellite cell by using an equine skeletal muscle satellite cell culture method to obtain an equine skeletal muscle satellite cell, and purifying by using a differential adherence method to obtain a purified equine skeletal muscle satellite cell;
step two, preparing a new culture dish, paving 2ml of gelatin solution in the culture dish, and putting the culture dish into the culture dish at 37 ℃ and 5% CO2Taking out the culture dish after 1-3 hours in the incubator, sucking out excessive gelatin, cleaning twice by using DPBS (double DPBS) to obtain a culture container with gelatin layers formed on the surface, and adding equine skeletal muscle satellite cells and a proliferation culture solution.
Step three, adding the culture container of the horse skeletal muscle satellite cells and the proliferation culture solution at 37 ℃ and 5% CO2And (5) culturing, namely culturing the equine skeletal muscle satellite cells until the confluence rate is 70-80%, namely, when the equine skeletal muscle satellite cells are normally passaged to the fourth generation, thus obtaining the adherent culture equine skeletal muscle satellite cells.
Adherent culture of equine skeletal muscle satellite cells at 1, 4, and 8 hours of culture, respectively, was observed under a microscope as shown in fig. 1, wherein a: adhering the gelatin-free skeletal muscle satellite cells for 1 h; b: gelatin-free skeletal muscle satellite cells for 4 h; c: gelatin-free skeletal muscle satellite cells for 8 h; d: laying gelatin for skeletal muscle satellite cell adherence for 1 h; e: laying gelatin for skeletal muscle satellite cell adherence for 4 h; f: and (5) paving gelatin on skeletal muscle satellite cells for 8 h.
Example 2 preparation of equine skeletal muscle satellite cell oxidative stress culture model
The preparation method of the equine skeletal muscle satellite cell oxidative stress culture model provided in this example includes adding H with different concentrations to the adherent culture equine skeletal muscle satellite cells prepared in example 12O2Stimulating the culture medium to culture for 2, 4, 6, and 8 hr respectively, and adding H with different concentrations2O2Culture dish for adherent culture of horse skeletal muscle satellite cells by stimulating culture solution, placing in culture dish at 37 deg.C and 5% CO2Culturing in an incubator to obtain the horse skeletal muscle satellite cell oxidative stress culture model.
Wherein contains H2O2The preparation method of the culture solution comprises the following steps: 0.5ml of a solution containing 30% (V/V) H2O2Diluting to 4.5ml deionized water, and mixing to obtain 1000 mmol/L H2O2Stock solution of H2O2Preparing stock solution into H with different concentrations2O2Stock solution of H2O2The concentrations of the stock solutions were: 0mmol/L, 100mmol/L, 200mmol/L, 400mmol/L, 600mmol/L, 800mmol/L and 1000 mmol/L. Respectively taking H with different concentrations2O2Adding 15 μ l of stock solution into 14.985ml of culture solution for proliferation of skeletal muscle satellite cells to obtain H2O2The action concentrations are respectively 0 mu mol/L, 100 mu mol/L, 200 mu mol/L, 400 mu mol/L, 600 mu mol/L, 800 mu mol/L and 1000 mu mol/L, and the mixture is filtered by a 0.22 mu m filter after being uniformly mixed.
Test example 1 Effect of oxidative stimulation on apoptosis Rate of equine skeletal muscle satellite cells
When the equine skeletal muscle satellite cells cultured in the embodiment 2 of the invention reach 70% -80% confluence, H with different concentrations is added2O2The cell apoptosis rate of the cultured cells is measured by culturing the stimulated culture solution for different times. As shown in Table 1, H2O2Effect of treatment on apoptosis Rate, varying concentrations of H2O2The apoptosis rate of the cells at different time is treated, and the result shows that the apoptosis rate is changed along with H in the culture solution2O2The increase in concentration, and the increase in treatment time, also increased the rate of apoptosis.
TABLE 1
Test example 2 Effect of oxidative stimulation on ROS in equine skeletal muscle satellite cells
When the equine skeletal muscle satellite cells cultured in the embodiment 2 of the invention reach 70% -80% confluence, H with different concentrations is added2O2The culture medium was cultured for various periods of time to measure ROS in equine skeletal muscle satellite cells. As shown in the figure2, with H in the culture medium2O2The concentration increased and the treatment time increased, the peak of ROS shifted right-hand, the intracellular ROS content increased, and the results coincided with the equine skeletal muscle satellite apoptosis rate. Therefore, the optimal treatment condition for the equine skeletal muscle satellite cell oxidative stress culture model is determined by treating for 6h at 600 mu mol/L/L according to the apoptosis rate and the intracellular ROS content.
Although the invention has been described in detail above with reference to a general description and specific examples, it will be apparent to one skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (8)
1. A method for improving the growth speed of equine skeletal muscle satellite cell adherent culture, which is characterized by comprising the steps of adding a gelatin solution into a culture vessel, and adding 5% CO at 37 DEG C2Placing for 1-3h in the environment to obtain a culture container with gelatin layer formed on the surface;
transferring the equine skeletal muscle satellite cells to the culture container with gelatin layer formed on the surface, adding proliferation culture solution, 37 ℃ and 5% CO2Culturing in the environment for 1-3h to obtain adherent culture equine skeletal muscle satellite cells.
2. The method of increasing the growth rate of adherent culture of equine skeletal muscle satellite cells according to claim 1,
and removing excessive gelatin solution in the culture container with the gelatin layer formed on the surface, cleaning twice by using DPBS, and adding the proliferation culture solution.
3. The method of increasing the growth rate of adherent culture of equine skeletal muscle satellite cells according to claim 1,
the mass concentration of the gelatin solution is 0.0025 g/ml.
4. The method of increasing the growth rate of adherent culture of equine skeletal muscle satellite cells according to claim 1,
the proliferation solution was 20% fetal bovine serum, 1% penicillin streptomycin and amphotericin B, DMEM top-up.
5. An equine skeletal muscle satellite cell oxidative stress culture model which is characterized in that,
adding a culture medium containing H to anchorage-cultured equine skeletal muscle satellite cells prepared by the method of any one of claims 1 to 42O2The horse skeletal muscle satellite cell oxidative stress culture model is obtained by culturing in the stimulation culture solution.
6. The equine skeletal muscle satellite cell oxidative stress culture model of claim 5,
said compound containing H2O2In the stimulated culture solution of (1), H2O2Concentrate 600. mu. mol/L.
7. The equine skeletal muscle satellite cell oxidative stress culture model of claim 5,
the adherent culture equine skeletal muscle satellite cells are cultured in the culture container with the gelatin layering formed on the surface to reach 70% -80% confluence.
8. The equine skeletal muscle satellite cell oxidative stress culture model of claim 5,
the addition contains H2O2The time for culturing in the stimulated culture solution of (2) to (10) hours.
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| CN103160461A (en) * | 2013-04-08 | 2013-06-19 | 哈尔滨体育学院 | In-vitro separation culture method for skeleton satellite cells of excellent ice and snow athletes |
| CN105647857A (en) * | 2016-04-08 | 2016-06-08 | 安徽农业大学 | Separation and purification method for skeletal muscle satellite cells in goats |
| CN111004776A (en) * | 2020-01-07 | 2020-04-14 | 内蒙古农业大学 | Method for separating and culturing equine skeletal muscle satellite cells |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050096731A1 (en) * | 2002-07-11 | 2005-05-05 | Kareen Looi | Cell seeded expandable body |
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