A chicken adipocyte adipogenic differentiation medium, induction method and application thereof

Abstract

The invention discloses a chicken adipocyte adipogenic differentiation medium, an induction method and an application, and relates to the technical field of poultry cell differentiation. The chicken adipocyte adipogenic differentiation medium comprises a basal medium, and further comprises 5% by volume of fetal bovine serum, 100 U / mL of penicillin, 100 μg / mL of streptomycin, 50-150 μmol / L of EPA, 5-10 μg / mL of insulin, 15-20 mmol / L of HEPES and 2-4 mmol / L of glutamine. The chicken adipocyte adipogenic differentiation medium provided by the invention can make the adipogenic differentiation efficiency of chicken adipocytes reach 100%, and a specific amount of EPA addition and a specific amount of fetal bovine serum can promote adipogenic differentiation.

Description

Technical Field

[0001] The invention relates to the technical field of avian cell differentiation, and in particular to a chicken adipocyte adipogenic differentiation medium, an induction method and an application thereof. Background Art

[0002] Cell-cultured meat is a new type of meat product made by extracting animal stem cells, usually muscle and fat stem cells, and culturing them in vitro. The stem cells undergo myogenic or adipogenic differentiation to form muscle fibers and mature fat cells, which are then collected and processed. Cell-cultured meat technology has the potential to become a sustainable and environmentally friendly way of producing meat. It can reduce dependence on traditional breeding and slaughtering processes, reduce the consumption of natural resources, and reduce environmental problems associated with traditional animal husbandry. In addition, cell-cultured meat can also provide more flexibility, enabling people to produce customized meat products to meet different consumer needs and health preferences.

[0003] The fat tissue of poultry is the main energy reserve. Fat cells store excess energy, which can be used by the body when needed to maintain normal physiological functions. Fat has good insulating properties, which can reduce heat loss and help birds maintain a stable body temperature. Especially in winter or cold areas, fat can provide an extra layer of insulation to help birds resist the adverse effects of low temperatures.

[0004] Fat plays an important role in the taste and mouthfeel of poultry meat. Fat can give poultry meat a sense of fullness and softness in the mouthfeel, making it more juicy and delicious. The right amount of fat can improve the taste of poultry meat and make it more flavorful. Fat is the main carrier of fat-soluble vitamins (such as vitamins A, D, E and K). These vitamins play an important role in the growth, immune function and other physiological processes of poultry. The fat-soluble vitamins in adipose tissue can provide these nutrients to poultry.

[0005] Fat cells in cultured meat not only play a key role in texture, taste and oral experience, but also provide meat products with important nutritional properties such as energy, fatty acids and fat-soluble vitamins. These properties enable cultured meat to better simulate the taste and nutritional value of traditional meat.

[0006] The process of adipocyte differentiation is the process of adipogenesis. Adipocyte differentiation is a complex process involving the regulation of multiple cell signaling pathways and transcription factors. Preadipocytes undergo cell proliferation, that is, an increase in the number of cells, under appropriate environmental stimulation. During cell proliferation, cells need to go through specific cell cycle phases, including G1, S, G2, and M phases. These phases are controlled by a series of cell cycle regulatory proteins and signaling pathways to ensure that cells undergo DNA replication and cell division at the appropriate time. In the process of adipocyte differentiation, some specific transcription factors play a key regulatory role. Among them, the most important are transcription factors of the C / EBP (CCAAT / enhancer binding protein) family and the PPAR (peroxisome proliferator-activated receptor) family. They promote adipocyte differentiation and fat metabolism by interacting and regulating the expression of a series of downstream genes. In the late stage of adipocyte differentiation, mature adipocytes begin to synthesize and store fat. This includes processes such as fatty acid synthesis, glycerol phosphorylation, and lipid droplet formation. Mature adipocytes have abundant lipid droplets for storing fat and have an impact on energy metabolism and endocrine function.

[0007] Overall, adipocyte differentiation is a complex process involving the regulation of multiple cell signaling pathways and transcription factors. This process is crucial for the normal development and function of adipose tissue and is also closely related to energy metabolism and endocrine function.

[0008] Poultry fat is mainly synthesized in the liver, and the ability of in vitro adipocytes to synthesize from scratch is poor. Therefore, the cocktail method used in traditional mammals (induction reagents include: insulin, dexamethasone, IBMX) has a poor induction effect, and almost no fat droplets are produced. Studies have shown that the in vitro differentiation of poultry adipocytes requires the addition of exogenous fatty acids. Suitable exogenous fatty acids can provide the fat substances required by poultry adipocytes, promote their differentiation and fat droplet production. The most commonly used exogenous fatty acid is oleic acid. In addition, the formulation of the culture medium is crucial for the in vitro differentiation of poultry adipocytes. For the induction differentiation medium of chicken adipocyte precursor cells, oleic acid and fetal bovine serum (FBS) are usually added to the existing basal culture medium, but there are still problems of low differentiation efficiency and unstable differentiation efficiency. Summary of the invention

[0009] In view of the above problems in the prior art, the present invention provides a chicken adipocyte adipogenic differentiation medium and an induction method to solve the problems of low differentiation efficiency and unstable differentiation efficiency in the prior art chicken adipocyte adipogenic differentiation technology.

[0010] Eicosapentaenoic acid, English name: Eicosapentaenoic Acid, English abbreviation: EPA, is the main component of fish oil. EPA belongs to the Omega-3 series of polyunsaturated fatty acids. Omega-3 polyunsaturated fatty acids are essential fatty acids for the human body. The human body cannot synthesize them by itself and can only be ingested through external food. EPA mainly comes from marine animals, such as fish, shrimp and other seafood, especially deep-sea fish.

[0011] The technical solution adopted by the present invention is as follows:

[0012] The invention discloses a chicken adipocyte adipogenesis induction differentiation culture medium. The chicken adipocyte adipogenesis induction differentiation culture medium comprises a basal culture medium, and further comprises 5% by volume of fetal bovine serum, 100 U / mL of penicillin, 100 μg / mL of streptomycin, 50-150 μmol / L of EPA, 5-10 μg / mL of insulin, 15-20 mmol / L of HEPES, and 2-4 mmol / L of glutamine.

[0013] The present invention provides a scheme for using EPA to replace traditional oleic acid as an adipogenic differentiation induction agent for chicken fat precursor cells. When used in combination with insulin at a relatively low fetal bovine serum concentration (5% by volume), the scheme can promote the formation of fat droplets in fat cells, thereby promoting the adipogenic differentiation of chicken fat precursor cells, so that the cell differentiation efficiency reaches 100%, and EPA, an essential fatty acid for human body, is added to chicken bio-cultured meat, thereby broadening the functionality of the bio-cultured meat.

[0014] Preferably, the basal culture medium is one of DMEM and DMEM / F12 culture medium.

[0015] Preferably, the concentration of EPA in the chicken adipocyte adipogenic differentiation medium is 150 μmol / L.

[0016] Preferably, the concentration of insulin in the chicken adipocyte adipogenic differentiation medium is 5 μg / mL.

[0017] The method for inducing cell differentiation using the chicken adipocyte adipogenesis induction differentiation medium comprises the following steps:

[0018] (1) chicken preadipocytes were obtained and cultured in vitro for proliferation until the confluence reached 80% or more, the proliferation medium was discarded, and chicken preadipocyte adipogenic differentiation medium was added, and the day of adding the differentiation medium was recorded as day 0;

[0019] (2) Inducing differentiation for 2 to 6 days to achieve adipogenic differentiation of chicken adipocytes. During the differentiation induction process, the differentiation medium is changed every 1 day.

[0020] The chicken fat precursor cell adipogenesis induction differentiation medium is used as a differentiation medium for seed cells in the process of preparing chicken biological culture meat.

[0021] The chicken fat precursor cell adipogenesis induction differentiation culture medium is used as a test reagent in the process of testing the differentiation performance of chicken seed cells using a cell differentiation method.

[0022] The chicken fat precursor cell adipogenesis induction differentiation medium is used as a research reagent in the process of studying the chicken fat development mechanism by utilizing in vitro cell differentiation.

[0023] In summary, compared with the prior art, the present invention has the following advantages and beneficial effects:

[0024] 1. The chicken adipocyte adipogenic differentiation medium provided by the present invention has good adipogenic differentiation effect. Experimental studies have shown that after the adipocytes are induced to differentiate in vitro for 6 days using the medium provided by the present invention, almost all cells have lipid droplets, the cell differentiation efficiency reaches 100%, and the repeatability is good; and in the research of the present invention, it is found that a specific amount of EPA and a specific amount of fetal bovine serum can increase the synthesis of lipid droplets in adipocytes, increase the fat content of adipocytes, and thus promote adipogenic differentiation.

[0025] 2. The present invention provides an adipogenic differentiation medium and an induction method suitable for chicken fat precursor cells, which is of great significance for the study of the mechanism of chicken fat development and the preparation of cultured meat.

[0026] 3. The present invention provides a method for preparing cultured chicken meat containing EPA (an Omega-3 polyunsaturated fatty acid), thereby increasing the flavor and nutritional properties of the cultured meat. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The microscopic morphology of the chicken adipocytes extracted in Example 1 (the scale in the figure is 250 um);

[0028] Figure 2 This is the identification diagram of chicken adipocytes extracted in Example 1 (the scale in the figure is 100 um);

[0029] Figure 3 Bright field images of chicken adipocytes after adipogenic differentiation for 0, 2, 4 and 6 days (the scale bar in the figure is 50 μm);

[0030] Figure 4 The Oil Red O staining images of chicken adipocytes after 0, 2, 4 and 6 days of adipogenic differentiation (the scale bar in the figure is 50 um);

[0031] Figure 5 BODIPY staining images of chicken adipocytes after 0, 2, 4 and 6 days of adipogenic differentiation (the scale bar in the figure is 100um);

[0032] Figure 6 The bright field images of chicken adipocytes after 2 and 4 days of adipogenic differentiation induction under the conditions of 2% fetal bovine serum, 5 μg / mL insulin concentration, and different EPA concentrations (50 μmol / L, 75 μmol / L, 100 μmol / L, 150 μmol / L) (the scale bar in the figure is 50 μm);

[0033] Figure 7 The bright field images of chicken adipocytes after 2 and 4 days of adipogenic differentiation induced by treatment with 5% fetal bovine serum, 5 μg / mL insulin concentration, and different EPA concentrations (50 μmol / L, 75 μmol / L, 100 μmol / L, and 150 μmol / L) (the scale bar in the figure is 50 μm);

[0034] Figure 8 Bright field images of chicken adipocytes after 2 and 4 days of adipogenic differentiation induction when the volume percentage of fetal bovine serum was 10%, the insulin concentration was 5 μg / mL, and the EPA concentrations were different (50 μmol / L, 75 μmol / L, 100 μmol / L, 150 μmol / L). The scale bar in the figure is 50 um. DETAILED DESCRIPTION

[0035] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0036] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0037] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0038] The present application will be described in detail below with reference to embodiments and experimental data.

[0039] Example 1

[0040] This example explores the separation and identification of chicken adipocytes for subsequent adipogenic differentiation experiments. The specific operations are as follows:

[0041] 1. Extract chicken adipocytes: Take subcutaneous adipose tissue from one-day-old chickens and cut into 1 mm 3 Add 3-4 times the volume of 2 mg / mL collagenase I to the fragments of different sizes, and digest them in a 37°C incubator for 30-40 min. Mix them by inverting every 5 min. When the tissue is digested to a quicksand state, add an equal volume of complete medium containing 20% ​​FBS to terminate the digestion. Filter with a 100 μm disposable cell sieve and centrifuge. The white precipitate at the bottom is the extracted cells. The microscopic morphology of the extracted cells is as follows: Figure 1 As shown, the extracted cells were preliminarily identified as chicken adipocytes; the extracted cells were added to complete culture medium to resuspend the cell pellet, and after counting, they were spread into cell culture dishes for subsequent proliferation culture.

[0042] 2. Identification of chicken preadipocytes: The specific marker of preadipocytes is preadipocyte factor-1 (delta like non-canonical Notch ligand 1, DLK1). Therefore, cell immunofluorescence technology was used to identify chicken preadipocytes using DLK1 as the primary antibody. The specific steps are as follows:

[0043] (1) Inoculate cells into 24-well culture plates;

[0044] (2) When the cell confluence reaches about 70%, discard the culture medium and wash the cells three times with PBS. Add 500 μL of cell fixative to the wells and fix for 20 to 30 minutes. Discard the fixative and wash the cells three times with PBS, 5 minutes each time.

[0045] (3) Add 0.1% Triton X-100 to permeabilize the cells for 15 to 20 min, discard the permeabilization solution, and wash with PBS three times, 5 min each time.

[0046] (4) Add 500 μL of 0.2% BSA and block at room temperature for 45 to 60 minutes;

[0047] (5) Discard the blocking solution, add 200 μL of rabbit DLK1 primary antibody (dilution ratio 1:50-1:100), and place at 4°C overnight. Wash with PBS three times, 5 min each time;

[0048] (6) Add 200 μL of goat anti-rabbit fluorescent secondary antibody (dilution ratio 1:100-1:200) and incubate at 4°C overnight;

[0049] (7) Wash with PBS three times, 5 min each time, and add 200 μL DAPI (1-2 μg / mL) staining solution to stain the cell nuclei;

[0050] (8) Wash with PBS three times, 5 min each time, and then observe and photograph using a fluorescence microscope.

[0051] The results are as follows Figure 2 As shown, the obtained chicken preadipocytes were identified by immunofluorescence technique, and it was found that almost all cells expressed DLK1 protein, indicating that the chicken preadipocytes were successfully isolated and obtained in this example.

[0052] Example 2

[0053] The chicken fat precursor cells extracted in Example 1 were subjected to adipogenic differentiation induction, and the specific operation was as follows:

[0054] 1. Preparation of adipogenic differentiation medium

[0055] Using DMEM / F12 medium as the basal medium, the following were added to the basal medium: fetal bovine serum, a mixture of penicillin and streptomycin, EPA, insulin, HEPES (4-hydroxyethylpiperazineethanesulfonic acid), and glutamine. After addition, the fetal bovine serum accounted for 5% of the volume of the culture medium, the concentration of penicillin was 100U / mL, the concentration of streptomycin was 100μg / mL, and the concentrations of EPA, insulin, HEPES, and glutamine were: EPA concentration was 150μmol / L, insulin concentration was 5μg / mL, HEPES concentration was 15mmol / L, and glutamine concentration was 2.5mmol / L.

[0056] Wherein, penicillin and streptomycin are added in the form of a penicillin-streptomycin mixture, which is a commercially available penicillin-streptomycin mixture (100×), in which the content of penicillin is 10 kU / mL and the content of streptomycin is 10 mg / mL. When added to the basal culture medium, the penicillin-streptomycin mixture accounts for 1% of the total culture medium volume.

[0057] 2. Adipogenic differentiation culture

[0058] The chicken fat precursor cells extracted in Example 1 were taken and cultured in vitro for proliferation until the confluence was more than 80%. The proliferation medium was removed, and the amplified chicken fat precursor cells were added to the adipogenic differentiation medium. The day of adding the differentiation medium was recorded as 0 day. The chicken adipogenic differentiation was achieved after 6 days of induction differentiation. During the differentiation induction process, the differentiation medium was changed every 1 day, and the differentiation bright field images were recorded every 1 day. The effect of differentiation for 6 days is shown in FIG. Figure 3 shown.

[0059] In order to further test the differentiation effect, this example also used Oil Red O staining and BODIPY staining to perform synchronous detection at different times of differentiation induction.

[0060] 1. Test the differentiation effect by Oil Red O staining

[0061] (1) Remove the adipogenic differentiation medium and wash with PBS 1 to 3 times;

[0062] (2) Add 4% PFA to fix the cells for 30 min.

[0063] (3) Remove the fixative solution and add the staining solution to soak for 30 seconds.

[0064] (4) Remove the washing solution and add Oil Red O staining solution to stain for 15 to 20 minutes.

[0065] (5) Discard the Oil Red O stain and wash 1 to 3 times with PBS;

[0066] (6) Observe under a microscope and take photos.

[0067] In the Oil Red O staining method, if an orange-red staining result appears, it means that lipid droplets are present. Figure 4 As shown in the figure, the results of Oil Red O staining show that after 2 days of adipogenic differentiation medium treatment, staining results begin to appear, indicating that lipid droplets begin to appear in the cells. As the differentiation time increases, the staining results increase, indicating that the content of lipid droplets also gradually increases. After 6 days of differentiation, the staining results almost fill the field of view, indicating that the cell differentiation efficiency is almost 100% at this time.

[0068] 2. Test the differentiation effect by the following BODIPY staining steps:

[0069] (1) Remove the adipogenic differentiation medium and gently rinse with PBS three times;

[0070] (2) Add 4% PFA to fix the cells for 20 min;

[0071] (3) Add 0.1% PBSTr (0.1% Triton X-100 prepared in PBS) to permeabilize the cells for 15 min;

[0072] (4) Remove the permeabilization solution and rinse once with PBS;

[0073] (5) Add 5 μmol / L BODIPY staining solution and 1.5 μg / mL DAPI staining solution and stain for 15 min in the dark;

[0074] (6) Discard the staining solution and rinse with PBS three times;

[0075] (7) Add anti-fluorescence quenching agent and observe and take pictures using a fluorescence microscope.

[0076] In the BODIPY staining method, if green staining results appear, it means that lipid droplets are present. Figure 5 As shown, the BODIPY staining results showed that: when treated with adipogenic differentiation medium for 0 days, the cells were not differentiated and no lipid droplets appeared, so the background color was blue and no green staining appeared; after 2 days of treatment, green staining began to appear, indicating that lipid droplets began to appear in the cells; as the differentiation time increased, the green staining portion increased, indicating that the lipid droplet content also gradually increased, especially after 6 days of treatment, the green staining almost filled the field of view, indicating that the cell differentiation efficiency was almost 100% at this time.

[0077] Combination Figure 3 to Figure 5 It is not difficult to see that lipid droplets appeared 2 days after induction of differentiation, and after 6 days of induction of differentiation, lipid droplets appeared in almost all cells, and the cell differentiation efficiency reached 100%.

[0078] Example 3

[0079] In this example, based on the culture medium in Example 2, the insulin concentration was 5 μg / mL, and the dosage of fetal bovine serum and EPA was used as two variables to further explore the effects of different dosages of fetal bovine serum and EPA on cell differentiation during the induction differentiation of chicken adipocytes. The specific experimental settings were:

[0080] (1) Variable 1: The volume percentage of fetal bovine serum was set to 2%, 5% and 10% respectively.

[0081] (2) Variable 2: The concentrations of EPA were set to 50 μmol / L, 75 μmol / L, 100 μmol / L, and 150 μmol / L, respectively.

[0082] (3) Constant: The insulin concentration was set to 5 μg / mL.

[0083] Specific induction differentiation operation: Take the chicken fat precursor cells extracted in Example 1, and proliferate and culture them in vitro until the confluence is more than 80%, remove the proliferation medium, add the amplified chicken fat precursor cells into the adipogenic induction differentiation medium, and record the day of adding the differentiation medium as 0d, and record the differentiation bright field images at 2d and 4d.

[0084] like Figure 6 As shown, when the insulin concentration was 5 μg / mL and the volume percentage of fetal bovine serum was 2%, both the 2d and 4d differentiation bright field images showed that EPA had no obvious promoting effect on cell differentiation, and higher concentrations of EPA would also have a lethal effect on chicken adipocytes.

[0085] like Figure 7 As shown, when the insulin concentration was 5 μg / mL and the volume percentage of fetal bovine serum was 5%, EPA could promote the adipogenic differentiation of chicken adipocytes in a dose-dependent manner.

[0086] like Figure 8 As shown, when the insulin concentration is 5 μg / mL and the volume percentage of fetal bovine serum is 10%, EPA can promote the differentiation of chicken adipocytes, but at the same EPA dose, its cell differentiation efficiency is lower than that at a serum concentration of 5%.

[0087] The above-mentioned embodiments only express the specific implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the protection scope of the present application. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the technical solution concept of the present application, and these all belong to the protection scope of the present application.

Claims

1. A chicken adipocyte adipogenesis induction differentiation medium, comprising a basal medium, characterized in that: It also includes 5% fetal bovine serum, 100 U / mL penicillin, 100 μg / mL streptomycin, 150 μmol / L EPA, 5 μg / mL insulin, 15-20 mmol / L HEPES, and 2-4 mmol / L glutamine.

2. The chicken adipocyte adipogenesis differentiation medium according to claim 1, wherein The basic culture medium is one of DMEM and DMEM / F12 culture medium.

3. A method for inducing cell differentiation using the chicken adipocyte adipogenesis induction differentiation medium according to claim 1 or 2, characterized in that: The steps include: (1) Take chicken preadipocytes and culture them in vitro until the confluence is above 80%. Discard the proliferation medium and add chicken preadipocyte adipogenic differentiation medium. The day of adding differentiation medium is recorded as day 0. (2) Induce differentiation for 2 to 6 days to achieve adipogenic differentiation of chicken fat precursor cells. During the differentiation induction process, change the differentiation medium every 1 day.

4. Use of the chicken fat precursor cell adipogenesis induction differentiation medium as claimed in claim 1 or 2 as a differentiation medium for seed cells in the process of preparing chicken biological cultivation meat.

5. The chicken adipocyte adipogenic differentiation medium as claimed in claim 1 or 2 is used as a test reagent in the process of testing the differentiation performance of chicken seed cells by cell differentiation method.

6. The chicken adipocyte adipogenic differentiation medium as claimed in claim 1 or 2 is used as a research reagent in a process of studying the chicken fat development mechanism by in vitro cell differentiation.

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