A method for separating, identifying and adipogenically differentiating mammalian adipocyte precursor cells, a composition and application thereof

Abstract

The present invention belongs to the technical field of in vitro cell differentiation. The present invention provides a method, composition and application for separation, identification and adipogenic differentiation of mammalian adipocytes. The mechanical method combined with collagenase digestion method is used to successfully separate and obtain mammalian adipocytes. Immunofluorescence technology is used to identify the obtained adipocytes. The adipogenic differentiation method of adipocytes is explored by a combination of multiple adipogenic induction factors. The differentiated mature adipocytes are identified by Oil Red O staining, BODIPY staining, triglyceride (TAG) content determination and / or Western blot technology. The results show that the adipogenic differentiation method of the present invention has good beneficial effects, is an ideal method for studying adipocyte differentiation, and has important application value in the fields of animal husbandry, biomedicine and biological meat cultivation.

Description

Technical Field

[0001] The present invention belongs to the technical field of in vitro cell differentiation, and in particular relates to a method for separation, identification and adipogenic differentiation of mammalian fat precursor cells, a composition and an application thereof. Background Art

[0002] As people pay more attention to sustainable food and animal welfare, bio-cultured meat as a potential alternative has received more and more attention. Bio-cultured meat, also known as cultured meat, cell-cultured meat, clean meat, etc., is a new type of meat food that uses animal cells cultured in vitro to control their rapid proliferation, directed differentiation, and collection and processing. The development of animal adipose tissue and the differentiation of fat cells are of great significance to meat quality and disease research.

[0003] In traditional animal husbandry, the quality and fat content of pork are usually affected by pig breeds and feeding conditions. By using adipocyte differentiation technology, the development process of pig adipocytes can be more accurately controlled and regulated, thereby achieving fine regulation of meat quality, which helps to improve the consistency and quality of meat and meet the market demand for different types of pork. For example, by promoting the differentiation of pig fat precursor cells, the fat content in pork can be increased and the quality of meat can be improved, making it more suitable for consumers' taste needs, so that high-quality meat cells can be produced under in vitro conditions, providing a basis for the production of biologically cultivated meat.

[0004] At present, there are some methods for promoting the differentiation of porcine adipocytes, such as culture medium compositions, gene regulation methods and small molecule compounds. However, these methods have some limitations, such as stability, low efficiency and side effects. For example, the commonly used method for mammalian adipocyte differentiation is the cocktail method, which is a hormone mixture of insulin (INS), dexamethasone (DEX) and 3-isobutyl-1-methylxanthine (IBMX). However, this method has low differentiation efficiency, uneven differentiation and poor stability. Therefore, a more effective and feasible method is needed to overcome these technical problems and promote the differentiation and maturation of porcine adipocyte precursors.

[0005] The specification of Chinese invention CN202210418352.3 records a method for preparing yak preadipocytes, in which the perirenal and subcutaneous fat of yak calves within one week of birth are digested in vitro by type I collagenase for primary culture, low-concentration trypsin digestion and microscopic observation combined with physical shaking for subculture, and modified induced differentiation medium for induced differentiation culture, and then mature yak adipocytes are obtained. This patent obtains good cell morphology, fast cell proliferation, and efficient induced differentiation treatment. It is a more effective method for culturing preadipocytes, providing a necessary carrier for studying the molecular mechanism of yak body fat deposition.

[0006] The above methods have certain effects, but the differentiation efficiency, differentiation uniformity and stability still need to be improved, and separation and differentiation methods with fewer side effects are also needed at present. Summary of the invention

[0007] In order to solve the above technical problems, the present invention provides a method, composition and application for isolating, identifying and adipogenically differentiating mammalian fat precursor cells, which have high differentiation efficiency, uniform differentiation, good stability and few side effects; the operation method is simple and efficient to achieve the differentiation and maturation of pig fat cells.

[0008] A method for separating, identifying and adipogenically differentiating mammalian adipocyte precursor cells to solve the above technical problems comprises the following steps:

[0009] Step 1: Isolation of mammalian adipocytes: using mechanical method combined with collagenase digestion;

[0010] Step 2: Identification of mammalian adipocyte precursor cells: using cell immunofluorescence method;

[0011] Step 2: promoting adipogenic induction of mammalian adipocytes in vitro differentiation: inducing adipogenic differentiation of adipocytes into mature adipocytes by a combination of multiple adipogenic induction factors;

[0012] Step 4. Identification of mature mammalian adipocytes: Identification is performed using Oil Red O staining, BODIPY staining, TAG (triglyceride) content determination and / or Western blot technology.

[0013] The mammals are humans and other mammals, preferably other mammals are pigs, cows, horses, sheep, mice and the like.

[0014] In the optimized scheme, the separation of mammalian fat precursor cells in step 1 specifically includes the following steps:

[0015] (1) Obtain subcutaneous fat from a mammal, cut it into minced meat, add 1-3 mg / mL collagenase solution, transfer it to a tissue dissociation tube, dissociate it using a tissue dissociator, place it in a 37°C water bath, digest it for 60-90 minutes, and dissociate it using a tissue dissociator every 15-20 minutes; then add an equal volume of high-glucose DMEM containing 10% fetal bovine serum to terminate the digestion, and filter it with a 70-150 μm cell sieve; preferably, filter it with a 100 μm cell sieve; the volume ratio of the minced meat to the collagenase solution is 1:5; wherein the collagenase is type I collagenase, and the mass concentration of the type I collagenase solution is 2 mg / mL;

[0016] (2) centrifuging the digestion solution at 800-1500 rpm at room temperature for 5-10 min, discarding the supernatant, and retaining the cell pellet; preferably, the digestion solution is centrifuged at 1000 rpm at room temperature for 8 min;

[0017] (3) adding 3-5 times the volume of red blood cell lysis buffer to the cell pellet to resuspend it, lyse it at room temperature for 5-10 minutes, then centrifuge it, discard the supernatant, and retain the cell pellet; preferably, at room temperature without ice bath;

[0018] (4) Resuspend the cells in high-glucose DMEM containing 20% ​​FBS and inoculate them into cell culture dishes. After subculturing, use high-glucose DMEM containing 10% FBS as the complete culture medium.

[0019] The induction period of the method of the present invention is 8 days, and lipid droplets begin to appear from the second day, and then the lipid droplets gradually increase and grow larger.

[0020] In the optimized scheme, the step 2 of the present invention includes using immunofluorescence technology to identify the fat precursor cells obtained in the step 1; preferably, using PREF1 as a primary antibody to perform cell immunofluorescence to characterize the fat precursor cells in the step 1; further preferably, specifically including the following steps:

[0021] (1) On the first day, cells were seeded into a 24-well cell culture plate;

[0022] (2) On the second day, discard the culture medium, wash with PBS, add 300-500 μL of cell fixative, fix for 10-30 min, discard the fixative, and wash with PBS 2-4 times;

[0023] (3) Add 0.1%-1% PBSTr to permeabilize the cells for 15-20 min, discard the PBSTr, and wash with PBS 2-4 times;

[0024] (4) Add 300-500 μL 0.2%-2% BSA and block at room temperature for 45-60 minutes;

[0025] (5) Discard the blocking solution, add 100-200 μL PREF1 primary antibody (rabbit source) (dilution ratio 1:50-1:100), place at 4°C overnight, and wash 2-4 times with PBS;

[0026] (6) Add 100-200 μL goat anti-rabbit secondary antibody (dilution ratio 1:100-1:200) and incubate at room temperature for 1-2 hours;

[0027] (7) Wash with PBS 2-4 times and add 100-200 μL of 1-5 μg / mL DAPI staining solution to stain the cell nuclei;

[0028] (8) After washing with PBS 2-4 times, observe and take pictures using a fluorescence microscope.

[0029] The step three specifically includes the following steps: adding a specific small molecule compound to the cell culture medium to directly promote the maturation of adipocytes:

[0030] (1) Inoculate cells into 6-well plates for 2D culture; or inoculate cells into roller bottles for 3D culture. 5 cells / mL culture medium, 1 microcarrier / 10mL culture medium, the speed was set at 25 rpm for 10 min; 0 rpm for 50 min, 24 cycles, and then the speed was set at 30 rpm for three-dimensional culture;

[0031] (2) After the cells have grown to full size (the confluence reaches 100%), continue culturing for 2 days;

[0032] (3) Replace the complete culture medium with adipogenic differentiation medium and induce differentiation for 2 days;

[0033] (4) Change to differentiation maintenance medium and maintain differentiation culture for 2 days;

[0034] (5) Change to complete medium and continue culturing for 4 days, changing the medium every 2 days;

[0035] The components of the adipogenic differentiation medium include high-glucose DMEM, insulin, glucocorticoids, IBMX, thiazolidinediones, sodium fatty acids and 2-10% fetal bovine serum; the components of the maintenance differentiation medium include high-glucose DMEM, insulin, sodium fatty acids, thiazolidinediones and 2-10% FBS; the complete culture medium is high-glucose DMEM with 10% FBS; preferably, the thiazolidinedione drug is one of troglitazone and rosiglitazone, and / or the sodium fatty acids are sodium oleate, and / or the glucocorticoids are one of dexamethasone and hydrocortisone.

[0036] The induction period of the method of the present invention is 8 days, and lipid droplets begin to appear from the second day, and then the lipid droplets gradually increase and grow larger.

[0037] In a further optimized solution, the method of the present invention identifies the mature adipocytes differentiated in step (iv) by Oil Red O staining, BODIPY staining, TAG content determination and / or Western blot technology; preferably, the differentiated adipocytes are collected, the total cell protein is extracted, and the expression of differentiation marker proteins ADIPOQ, FABP4, PPARγ, CEBPα is detected by Western blot technology, and TUBB and ACTB are used as internal references at the same time; preferably, BODIPY staining is used, comprising the following steps:

[0038] (1) Discard the culture medium and rinse 1-2 times with PBS;

[0039] (2) Add cell fixative, fix for 10-30 min, and rinse with PBS 2-4 times;

[0040] (3) Add 1-5 μmol / L BODIPY working solution and 1-5 μg / mL DAPI solution and stain for 10-15 minutes in the dark;

[0041] (4) Discard the staining solution and rinse with PBS three times;

[0042] (5) Observe and take photos using a fluorescence microscope.

[0043] The present invention discloses a composition for separation, identification and adipogenic differentiation of mammalian adipocytes, comprising insulin, IBMX, glucocorticoids, sodium fatty acids and thiazolidinediones; preferably, the thiazolidinediones are one of troglitazone and rosiglitazone (RSG), and / or the sodium fatty acids are sodium oleate, and / or the glucocorticoids are one of dexamethasone (DEX) and hydrocortisone; or the composition consists of insulin, IBMX, dexamethasone, sodium oleate and rosiglitazone.

[0044] Insulin can promote glucose in the blood circulation to enter liver cells, muscle cells, fat cells and other tissue cells to synthesize glycogen, thereby lowering blood sugar and promoting the synthesis of fat and protein.

[0045] Glucocorticoids are an extremely important class of regulatory molecules in the body. They play an important regulatory role in the body's development, growth, metabolism, and immune function. They are the most important regulatory hormones for the body's stress response. They have anti-inflammatory, anti-toxic, anti-allergic, anti-shock, and non-specific immune suppression effects.

[0046] IBMX is a phosphodiesterase (PDE) inhibitor, also known as 3-isobutyl-1-methylxanthine. It inhibits the cleavage of cAMP by inhibiting phosphodiesterase F-III, thereby increasing the concentration of intracellular cAMP, increasing the influx of calcium ions, and producing a positive inotropic effect. In addition to the positive inotropic effect, phosphodiesterase inhibitors also have a vasodilatory effect by increasing the content of cAMP in vascular smooth muscle cells.

[0047] Rosiglitazone can activate PPAR-γ nuclear receptor and participate in the regulation of fatty acid metabolism.

[0048] Troglitazone, also known as Rezorlin, can regulate adipocyte differentiation and lipid metabolism.

[0049] Sodium fatty acid, also known as sodium octadecenoate, is a compound composed of a hydrophobic group and a hydrophilic group, used as an anionic surfactant. It is also a raw material for the synthesis of triglycerides.

[0050] The invention discloses an adipogenic differentiation medium for promoting differentiation of porcine adipocyte precursor cells. The medium comprises a basal medium high-glucose DMEM, which contains, based on the volume of the basal medium high-glucose DMEM, 5-10 μg / mL insulin, 0.2-0.8 μg / mL dexamethasone, 0.3-0.6 mmol / L IBMX, 1-5 μmol / L rosiglitazone, 100-200 μmol / L sodium oleate and 2-10% fetal bovine serum; preferably, 5 μg / mL insulin, 0.4 μg / mL dexamethasone, 0.5 mmol / L IBMX, 1 μmol / L thiazolidinedione, 150 μmol / L sodium fatty acid and 2-10% fetal bovine serum; preferably, 10% fetal bovine serum.

[0051] The present invention discloses a maintenance differentiation medium for promoting differentiation of pig adipocyte precursor cells. The medium comprises a basal medium high-glucose DMEM, which contains, based on the volume of the basal medium high-glucose DMEM, 5-10 μg / mL insulin, 1-5 μmol / L rosiglitazone, 100-200 μmol / L sodium oleate and 2-10% fetal bovine serum; preferably, 5 μg / mL insulin, 1 μmol / L thiazolidinedione, 150 μmol / L sodium fatty acid and 2-10% fetal bovine serum; preferably, 10% fetal bovine serum.

[0052] The application of the culture medium in the present invention is the application of the adipogenic differentiation culture medium and the maintenance differentiation culture medium, comprising the following steps:

[0053] (1) When the confluence of mammalian adipocytes reaches 100%, continue culturing for 2 days;

[0054] (2) Discard the old culture medium and add adipogenic differentiation medium to induce differentiation for 2 days;

[0055] (3) Discard the adipogenic differentiation medium and add the differentiation maintenance medium to maintain differentiation for 2 days;

[0056] (4) Discard the differentiation maintenance medium, add complete medium, and continue culturing for 4 days, changing the medium every 2 days;

[0057] (5) Observe and take photos using a microscope.

[0058] The application of the composition of the present invention is the application in the preparation of a medicine or composition for preventing, alleviating and / or treating lipodystrophy, a medicine for regulating fat development and metabolism, and an application in a culture medium for adipogenic differentiation; wherein the active ingredients are insulin, glucocorticoids, IBMX, thiazolidinediones and sodium fatty acids; preferably, the thiazolidinediones are one of troglitazone and rosiglitazone, and / or the sodium fatty acids are sodium oleate, and / or the glucocorticoids are one of dexamethasone and hydrocortisone.

[0059] The invention is further applied in the preparation of a kit for preparing an adipocyte adipogenic differentiation additive and a kit for improving an animal model of lipodystrophy.

[0060] The application of the adipogenic differentiation culture medium comprises a basic culture medium high-glucose DMEM, fetal bovine serum, insulin, dexamethasone, IBMX, rosiglitazone and sodium oleate.

[0061] The effective components of the prepared medicine are a combination of insulin, dexamethasone, IBMX, rosiglitazone and sodium oleate.

[0062] The methods, compositions and applications of the present invention can achieve the differentiation and maturation of mammalian fat cells under simpler and more efficient conditions; the differentiation efficiency is high, the differentiation is uniform and stable, and the side effects are few; whether in the ordinary culture method such as two-dimensional culture or in three-dimensional culture, there are very good differentiation effects; better tools and methods can be provided for animal husbandry, biomedical research and biologically cultivated meat research, and further understanding of the development process of mammalian fat cells can be used to regulate fat development and metabolism, as well as physiological and pathological processes related thereto. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 This is a cell morphology diagram of the porcine adipocytes separated in Example 5 of the present invention;

[0064] Figure 2 This is a diagram showing the results of immunofluorescence identification of PREF1 in porcine adipocytes in Example 5 of the present invention (Note: blue represents the cell nucleus and red represents the PREF1 protein);

[0065] Figure 3 This is an identification diagram of pig mature adipocytes in Example 7 of the present invention;

[0066] Figure 4 This is a graph showing the results of determining the triglyceride content of pig adipocytes differentiated on day 0 and day 8 in Example 7 of the present invention (***p<0.001 indicates an extremely significant difference);

[0067] Figure 5 This is a diagram showing the expression detection results of adipogenic differentiation-related genes in Example 7 of the present invention;

[0068] Figure 6 This is a diagram showing the results of lipid droplet staining during adipogenic differentiation of pig adipocytes in three-dimensional culture in Example 8 of the present invention (Scale bar: 100 μm);

[0069] Figure 7 and Figure 8 This is a comparison chart of the differentiation results of 8 different adipogenic differentiation compositions in Example 6 of the present invention. DETAILED DESCRIPTION

[0070] The present invention is further described below in conjunction with specific embodiments:

[0071] Example 1 A composition for separation, identification and adipogenic differentiation of mammalian adipocytes, comprising insulin, IBMX, glucocorticoids, sodium fatty acid and thiazolidinediones;

[0072] In this embodiment, the thiazolidinedione drug is further selected from troglitazone and rosiglitazone (RSG), and / or the sodium fatty acid is sodium oleate, and / or the glucocorticoid is selected from dexamethasone (DEX) and hydrocortisone; or the composition consists of insulin, IBMX, glucocorticoid, sodium oleate and rosiglitazone.

[0073] Furthermore, the culture medium for promoting differentiation of adipocyte precursor cells is composed of the following components and amounts: a basic culture medium high-glucose DMEM containing 5 μg / mL insulin, 0.4 μg / mL dexamethasone, 0.5 mmol / L IBMX, 1 μmol / L rosiglitazone, 150 μmol / L sodium oleate and 2% fetal bovine serum.

[0074] The maintenance differentiation medium for promoting differentiation of porcine adipocytes is composed of the following components and amounts: a basal medium high-glucose DMEM, which contains 5 μg / mL insulin, 1 μmol / L rosiglitazone, 150 μmol / L sodium oleate and 2% fetal bovine serum based on the mass of the basal medium.

[0075] Example 2 A culture medium for the isolation, identification and adipogenic differentiation of mammalian preadipocytes, wherein the culture medium for promoting the differentiation of preadipocytes consists of the following components and amounts: a basal culture medium high-glucose DMEM, containing, based on the volume of the basal culture medium, 5 μg / mL insulin, 0.4 μg / mL dexamethasone, 0.5 mmol / L IBMX, 1 μmol / L rosiglitazone, 150 μmol / L sodium oleate and 10% fetal bovine serum.

[0076] The maintenance differentiation medium for promoting differentiation of porcine adipocytes is composed of the following components and amounts: a basal medium high-glucose DMEM containing 5 μg / mL insulin, 1 μmol / L rosiglitazone, 150 μmol / L sodium oleate and 10% fetal bovine serum based on the volume of the basal medium.

[0077] Example 3 An adipogenic differentiation medium for promoting differentiation of porcine adipocytes, wherein the medium comprises a basal medium high-glucose DMEM, which contains, based on the volume of the basal medium DMEM, 5 μg / mL insulin, 0.2 μg / mL dexamethasone, 0.3 mmol / L IBMX, 1 μmol / L rosiglitazone, 100 μmol / L sodium oleate and 8% fetal bovine serum.

[0078] A maintenance differentiation medium for promoting differentiation of porcine adipocyte precursor cells, the medium comprising a basal medium high-glucose DMEM, which contains 5 μg / mL insulin, 1 μmol / L rosiglitazone, 100 μmol / L sodium oleate and 8% fetal bovine serum based on the volume of the basal medium high-glucose DMEM.

[0079] Example 4 An adipogenic differentiation medium for promoting differentiation of porcine adipocytes, the medium comprising a basal medium high-glucose DMEM, which contains, based on the volume of the basal medium high-glucose DMEM, 10 μg / mL insulin, 0.8 μg / mL glucocorticoid, 0.6 mmol / L IBMX, 5 μmol / L rosiglitazone, 200 μmol / L sodium oleate and 5% fetal bovine serum.

[0080] A maintenance differentiation medium for promoting differentiation of porcine adipocyte precursor cells, the medium comprising a basal medium high-glucose DMEM, which contains 10 μg / mL insulin, 5 μmol / L rosiglitazone, 200 μmol / L sodium oleate and 5% fetal bovine serum based on the volume of the basal medium high-glucose DMEM.

[0081] Example 5 Methods for isolating, identifying and differentiating porcine adipocytes.

[0082] In this embodiment, ophthalmic scissors were used to cut the pig fat tissue into 1 mm 3 The minced meat was then digested with collagenase type I, and then passed through a cell sieve and centrifuged to obtain pig fat precursor cells. Under a microscope, it was found that pig fat precursor cells were irregular long spindles, triangles, etc. ( Figure 1 The obtained porcine preadipocytes were identified by cell immunofluorescence, and the results showed that almost all cells expressed PREF1 protein (a marker protein of preadipocytes), indicating that porcine preadipocytes were successfully isolated and obtained ( Figure 2 ), which provided materials for studying the in vitro differentiation of porcine adipocytes; the specific methods are as follows:

[0083] The steps for isolating porcine preadipocytes are as follows:

[0084] (1) The piglets were anesthetized and killed;

[0085] (2) Transfer the piglet to a biosafety cabinet that has been sterilized with ultraviolet light and wipe the entire body with 75% alcohol for disinfection;

[0086] (3) Use a sterile scalpel, ophthalmic scissors, and ophthalmic forceps to separate the subcutaneous fat of the neck;

[0087] (4) Rinse the subcutaneous fat in 75% alcohol and then transfer it to PBS solution;

[0088] (5) Remove the blood vessels, muscles and other impurities visible to the naked eye to obtain the subcutaneous fat of the pig, and cut the subcutaneous fat into pieces of about 1mm 3 Minced meat of different sizes;

[0089] (6) Add 5 volumes of 2 mg / mL type I collagenase solution to the centrifuge tube containing minced meat; transfer to the dissociation tube;

[0090] (7) After dissociation using a tissue dissociator, place the above dissociation tube in a 37°C water bath and digest for 60 minutes, dissociating once every 15 minutes using a tissue dissociator;

[0091] (8) Add an equal volume of complete culture medium (high-glucose DMEM containing 10% fetal bovine serum) to terminate digestion and filter with a 100 μm cell sieve;

[0092] (9) Centrifuge the digestion solution at 1000 rpm at room temperature for 8 min; discard the supernatant and retain the cell pellet;

[0093] (10) Add 3 times the volume of minced meat and red blood cell lysis buffer (ACK Lysis Buffer) to the cell pellet to resuspend it. Lyse it at room temperature for 10 min and then centrifuge it. Discard the supernatant and keep the cell pellet. Place it at room temperature without ice bath.

[0094] (11) Resuspend the cells in high-glucose DMEM containing 20% ​​FBS, count the cells, and seed them into a 10 cm cell culture dish at an appropriate cell density. After subculturing, use high-glucose DMEM containing 10% FBS as the complete culture medium.

[0095] Immunofluorescence technology is used to identify the fat precursor cells obtained in the step 1; PREF1 is used as a primary antibody to perform cell immunofluorescence to characterize the fat precursor cells in the step 1. The steps of immunofluorescence identification of pig fat precursor cells are as follows:

[0096] (1) On the first day, cells were seeded into a 24-well cell culture plate;

[0097] (2) On the second day, discard the culture medium and wash the cells three times with PBS. Add 300 μL of cell fixative to the wells and fix for 15 min. Discard the fixative and wash the cells three times with PBS, 5 min each time.

[0098] (3) Add 0.1% PBSTr to permeabilize the cells for 15 min, discard the PBSTr, and wash with PBS three times, 5 min each time;

[0099] (4) Add 300 μL of 0.2% BSA and block at room temperature for 60 min;

[0100] (5) Discard the blocking solution, add 200 μL of diluted (dilution ratio 1:50-1:100) PREF1 primary antibody (rabbit source), and place at 4°C overnight; wash with PBS three times, 5 min each time;

[0101] (6) Add 200 μL of diluted goat anti-rabbit secondary antibody (dilution ratio 1:100-1:200) and incubate at room temperature for 1 h;

[0102] (7) Wash with PBS three times, 5 min each time, then dilute 100 μL of 2 μg / ml DAPI to 1 μg / mL with PBS, and add 200 μL of staining solution to the wells for cell nucleus staining;

[0103] (8) Wash with PBS three times, 5 min each time, and then observe and photograph using a fluorescence microscope.

[0104] Example 6 Screening of methods for promoting adipogenic differentiation of preadipocytes

[0105] The steps of adipogenic differentiation of porcine adipocytes are as follows;

[0106] (1) Seeding cells into 6-well plates for two-dimensional culture;

[0107] (2) When the confluence of porcine adipocytes reaches 100%, continue culturing for 2 days;

[0108] (3) Replace the original complete culture medium with adipogenic differentiation medium and induce differentiation for 2 days;

[0109] (4) Change to differentiation maintenance medium and maintain differentiation culture for 2 days;

[0110] (5) Change to complete medium and continue culturing for 4 days, changing the medium every 2 days;

[0111] (6) Observe and take photos using an ordinary microscope.

[0112] The components of the adipogenic differentiation medium are high-glucose DMEM, insulin, dexamethasone, IBMX, rosiglitazone, sodium oleate and 2% or 10% fetal bovine serum; the components of the maintenance differentiation medium are high-glucose DMEM, insulin, sodium oleate, rosiglitazone and 2% or 10% FBS; the complete culture medium is high-glucose DMEM with 10% FBS.

[0113] In this embodiment, under the same other conditions, four combinations of adipocyte inducers and two serum concentrations were compared, namely: (1) Cocktail, sodium oleate and 2% FBS; (2) Cocktail, sodium oleate, RSG (rosiglitazone) and 2% FBS; (3) Cocktail and 2% FBS; (4) Cocktail, RSG and 2% FBS; (5) Cocktail, sodium oleate and 10% FBS; (6) Cocktail, sodium oleate, RSG and 10% FBS; (7) Cocktail and 10% FBS; (8) Cocktail, RSG and 10% FBS.

[0114] The results are as follows Figure 7 and Figure 8 As shown, under the same induction reagent, high concentration of serum has a better effect on inducing differentiation, while under the same concentration of serum, the combination of cocktail method with sodium oleate and rosiglitazone has a better effect on cell differentiation. That is, the group of "Cocktail, sodium oleate, RSG 10% FBS" has the best differentiation effect.

[0115] Adipogenic differentiation medium was: high glucose DMEM + Cocktail + sodium oleate + RSG + 10% FBS; and maintenance differentiation medium was: high glucose DMEM + INS (insulin) + sodium oleate + RSG + 10% FBS. The adipogenic differentiation medium contained 5 μg / mL insulin, 0.4 μg / mL dexamethasone, 0.5 mmol / L IBMX, 1 μmol / L rosiglitazone, and 150 μmol / L sodium oleate; the maintenance differentiation medium contained 5 μg / mL insulin, 1 μmol / L rosiglitazone, and 150 μmol / L sodium oleate.

[0116] Example 7 Identification of mature adipocytes and expression of differentiation-related genes

[0117] Adipocyte staining methods generally include Oil Red O staining, BODIPY staining, etc. In addition, direct observation is also possible without staining.

[0118] Oil Red O is a fat-soluble dye that is highly soluble in fat and specifically colors neutral fats such as triglycerides in cells. BODIPY is a near-infrared short-wavelength fluorescent dye that specifically acts on oil droplets composed of neutral lipids.

[0119] In the present invention, direct observation, Oil Red O staining, and BODIPY staining were used to directly observe and stain cells at undifferentiated (0 d) and differentiated 8 d (using a cocktail, sodium oleate, and RSG in 10% FBS).

[0120] The results are as follows Figure 3 As shown, before staining, vacuolar lipid droplets could be observed in the cells differentiated for 8 days. After staining, these lipid droplets were stained bright red by Oil Red O and green by BODIPY, indicating that the adipogenic differentiation method using the "Cocktail, sodium oleate, RSG in 10% FBS" composition successfully promoted the adipogenic differentiation of porcine adipocytes into mature adipocytes.

[0121] The core component of lipid droplets is triglyceride. The present invention collects adipocytes differentiated on day 0 and day 8, and uses a cell triglyceride determination kit to determine the triglyceride content.

[0122] The results are as follows Figure 4 As shown in the figure, compared with undifferentiated adipocytes (0d), the content of triglyceride (TAG) in cells differentiated for 8d was significantly increased. For specific detection methods, see the Prilep Tissue Cell Triglyceride (TG) Content Enzymatic Assay Kit (Cat. No. E1013).

[0123] The present invention collects the adipocytes differentiated at 0d, 2d, 4d, 6d and 8d, extracts the total cell protein, and detects the expression of PREF1, ADIPOQ, FABP4, PPARγ and CEBPα by Western blot technology, and TUBB and ACTB are used as internal references at the same time.

[0124] The results are as follows Figure 5 As shown in the figure, the expression of DLK1 in preadipocytes gradually decreased from 2 days, and the expression was very weak in cells differentiated for 8 days, which was consistent with the expected results. However, the expression of ADIPOQ, FABP4, PPARγ, and CEBPα gradually increased with differentiation, and FABP4, as a marker of terminal differentiation, reached its peak on the 8th day of differentiation. Figure 4 The detection method is as follows:

[0125] Use mature adipocyte Oil Red O staining as follows:

[0126] (1) Discard the culture medium and gently rinse once with PBS;

[0127] (2) Add cell fixative, fix for 10 min, and rinse twice with PBS;

[0128] (3) Add an appropriate amount of staining and washing solution to cover the cells for 20 seconds;

[0129] (4) Discard the staining and washing solution, add an appropriate amount of modified Oil Red O staining solution, and stain for 15 minutes in the dark;

[0130] (5) Discard the modified Oil Red O staining solution, add an appropriate amount of staining and washing solution, and let it stand for 30 seconds;

[0131] (6) Discard the staining solution, wash with PBS for 20 seconds, observe under a microscope and take pictures.

[0132] Use BODIPY dye solution to dye, the steps are as follows:

[0133] (1) Discard the culture medium and rinse once with PBS;

[0134] (2) Add cell fixative, fix for 10 min, and rinse with PBS three times;

[0135] (3) Add 5 μmol / L BODIPY working solution and 5 μg / mL DAPI solution and stain for 10 min in the dark;

[0136] (4) Discard the staining solution and rinse with PBS three times, 5 min each time;

[0137] (5) Observe and take photos under a fluorescence microscope.

[0138] Cell protein extraction:

[0139] (1) Discard the culture medium and rinse once with PBS;

[0140] (2) Add an appropriate amount of cell lysis buffer containing protease inhibitors (300 μL per well of a 6-well plate) and place on ice for 10 min.

[0141] (3) Use a cell scraper to scrape the cells from the bottom of the well from top to bottom and from left to right, and mix them by pipetting;

[0142] (4) Collect the cell lysate into a 1.5 mL centrifuge tube and centrifuge at 12,000 rpm for 30 min at 4°C;

[0143] (5) Transfer the supernatant to a new 1.5 mL centrifuge tube. The supernatant is the protein and should be stored at -80°C until use.

[0144] Western blot:

[0145] (1) Take out the protein from the -80 ℃ freezer and place it on ice to thaw. Mix 20 μL of protein with 5 μL of 5× protein loading buffer and boil at 100 ℃ for 10 min to denature the protein.

[0146] (2) Prepare 10%-12% SDS-PAGE gel;

[0147] (3) Add 10 μL of denatured protein to each lane, and reserve one lane for 5 μL of prestained protein marker;

[0148] (4) Set the electrophoresis conditions to 120 V for 15 min and 200 V for 40 min. Terminate the electrophoresis when the bromophenol blue indicator band migrates to about 1 cm from the bottom of the gel.

[0149] (5) Using the wet transfer method, set a constant current of 400 mA and transfer to the PVDF membrane for about 30 minutes;

[0150] (6) Place the PVDF membrane in an antibody incubation box containing blocking solution at room temperature for 1 hour;

[0151] (7) Dilute the primary antibody at 1:5000 with blocking solution, add the primary antibody to the antibody incubation box, and incubate at 4°C overnight;

[0152] (8) Wash the membrane three times with TBST, 10 min each time;

[0153] (9) Dilute the secondary antibody at 1:5000 with TBST, add the secondary antibody to the antibody incubation box, and incubate at room temperature for 1 h;

[0154] (10) Wash the membrane three times with TBST, 10 min each time;

[0155] (11) Use ECL chemiluminescent liquid for detection and use chemiluminescent imaging system for observation and photography.

[0156] Example 8 Application of porcine adipogenic differentiation in three-dimensional culture

[0157] Compared with traditional two-dimensional culture, three-dimensional cell culture is closer to the physiological environment in vivo. Add the adipogenic differentiation medium containing "high-glucose DMEM, cocktail, sodium oleate, RSG and 10% FBS" to the three-dimensional culture flask, and replace it with the differentiation medium containing "high-glucose DMEM, INS, sodium oleate, RSG and 10% FBS" after 2 days and apply the differentiation method to three-dimensional culture. After BODIPY staining, it was found that the adipogenic differentiation of fat precursor cells into mature adipocytes can be successfully carried out in three-dimensional culture ( Figure 6 ). This indicates that the porcine adipocyte differentiation method of the present invention is applicable to two-dimensional and three-dimensional culture.

[0158] The specific method of three-dimensional culture and adipogenic differentiation of porcine adipocytes is as follows:

[0159] (1) Inoculate cells into roller bottles for 10 5cells / mL culture medium, 1 microcarrier / 10 mL culture medium, the speed was set to 25 rpm for 10 min; 0 rpm for 50 min, 24 cycles, and then the speed was set to 30 rpm;

[0160] (2) After the cells have grown to full size, continue culturing for 2 days;

[0161] (3) Replace the complete culture medium with adipogenic differentiation medium and induce differentiation for 2 days;

[0162] (4) Change to differentiation maintenance medium and maintain differentiation culture for 2 days;

[0163] (5) Change to complete medium and continue culturing for 4 days, changing the medium every 2 days;

[0164] The application of the above culture medium in the present invention lies in the application of the adipogenic differentiation medium and the maintenance differentiation medium, and the steps are as follows:

[0165] (1) When the confluence of animal adipocyte precursor cells reaches 100%, continue culturing for 2 days;

[0166] (2) Discard the old culture medium and add adipogenic differentiation medium to induce differentiation for 2 days;

[0167] (3) Discard the adipogenic differentiation medium and add the differentiation maintenance medium to maintain differentiation for 2 days;

[0168] (4) Discard the differentiation maintenance medium, add complete medium, and continue culturing for 4 days, changing the medium every 2 days;

[0169] (5) Observe and take photos using a microscope.

[0170] BODIPY staining of lipid droplets

[0171] (1) Pipette 1 mL of cell suspension, allow the cells and microcarriers to settle together, discard the supernatant medium, and rinse once with PBS.

[0172] (2) Add cell fixative, fix for 10 min, and rinse twice with PBS;

[0173] (3) Add an appropriate amount of 5 μmol / L BODIPY working solution and DAPI (to stain the cell nucleus at the same time) and cover the cells for staining in the dark for 10 min;

[0174] (4) Discard the staining solution and wash with PBS three times, 5 min each time;

[0175] (5) Use a high-content cell imaging system to scan and take pictures.

[0176] Example 9 The other contents are the same as those of Example 5, wherein: in the step of isolating porcine adipocytes:

[0177] (6) Add 5 volumes of 1 or 3 mg / mL type I collagenase solution to the centrifuge tube containing minced meat and transfer to the tissue dissociation tube;

[0178] (7) Dissociate the tissue dissociation tube using a tissue dissociator and place it in a 37°C water bath for 60 or 90 minutes, dissociating it every 20 minutes using a tissue dissociator;

[0179] (8) Add an equal volume of complete culture medium (high-glucose DMEM containing 10% fetal bovine serum) to terminate digestion and filter with a 70 or 150 μm cell sieve;

[0180] (9) Centrifuge the digestion solution at 800 or 1500 rpm at room temperature for 5 or 10 min; discard the supernatant and retain the cell pellet;

[0181] (10) Add 5 times the volume of minced meat red blood cell lysis buffer (ACK Lysis Buffer) to the cell pellet to resuspend it. Lyse at room temperature for 5 or 8 minutes and then centrifuge. Discard the supernatant and keep the cell pellet. Keep it at room temperature without ice bath.

[0182] (11) Resuspend the cells in high-glucose DMEM containing 20% ​​FBS, count the cells, and seed them into a 10 cm cell culture dish at an appropriate cell density. After subculturing, use high-glucose DMEM containing 10% FBS as the complete culture medium.

[0183] The steps of immunofluorescence identification of porcine adipocytes are as follows:

[0184] (1) On the first day, cells were seeded into a 24-well cell culture plate;

[0185] (2) On the second day, discard the culture medium and wash the cells three times with PBS. Add 400 or 500 μL of cell fixative to the wells and fix for 15 min. Discard the fixative and wash the cells 2 or 4 times with PBS, 5 min each time.

[0186] (3) Add 0.1-1% PBSTr to permeabilize the cells for 20 min, discard the PBSTr, and wash with PBS 2 or 4 times, 5 min each time;

[0187] (4) Add 400 or 500 μL of 0.2-2% BSA and block at room temperature for 45 or 50 min;

[0188] (5) Discard the blocking solution, add 100-200 μL of diluted (dilution ratio 1:50-1:100) PREF1 primary antibody (rabbit source), and place at 4°C overnight; wash with PBS 2 or 4 times, 5 min each time;

[0189] (6) Add 100-200 μL of diluted goat anti-rabbit secondary antibody (dilution ratio 1:1000-1:200) and incubate at room temperature for 2 h;

[0190] (7) Wash with PBS 2 or 4 times, 5 min each time, then dilute 150 or 200 μL of 1 or 5 μg / ml DAPI to 1 μg / mL with PBS, and add 200 μL of staining solution to the wells for nucleus staining;

[0191] (8) Wash with PBS 2 or 4 times, 5 min each time, and then observe and photograph using a fluorescence microscope.

[0192] The culture medium not mentioned by specific name is a complete culture medium, and the detection equipment used is conventional equipment or instrument.

[0193] The above implementation / test examples are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A method for inducing adipogenic differentiation of mammalian adipocytes, characterized in that: The steps of adipogenic differentiation are as follows: (1) Mammalian adipocytes were seeded into 6-well plates for two-dimensional culture; or adipocytes were seeded into roller bottles for 10 5 cells / mL culture medium, 1 microcarrier / 10 mL culture medium, the rotation speed is set to 25 rpm, 10 min; 0 rpm, 50 min, 24 cycles, and then the rotation speed is set to 30 rpm for three-dimensional culture, wherein the mammalian fat precursor cells are porcine fat precursor cells; (2) After the cells have grown to full size, continue culturing for 2 days; (3) The culture medium was changed to adipogenic differentiation medium and the differentiation was induced for 2 days; (4) Change the adipogenic differentiation medium to the differentiation maintenance medium and maintain differentiation culture for 2 days; (5) Replace the differentiation maintenance medium with complete medium and continue culturing for 4 days, changing the medium every 2 days; The adipogenic differentiation medium is based on 90% high glucose DMEM and 10% fetal bovine serum, with the addition of 5 μg / mL insulin, 0.4 μg / mL dexamethasone, 0.5 mmol / L IBMX, 1 μmol / L rosiglitazone, and 150 μmol / L sodium oleate; the maintenance differentiation medium is based on 90% high glucose DMEM and 10% fetal bovine serum, with the addition of 5 μg / mL insulin, 1 μmol / L rosiglitazone, and 150 μmol / L sodium oleate.

2. The method for adipogenic differentiation of mammalian adipocytes according to claim 1, characterized in that: The preparation steps of the mammalian fat precursor cells are as follows: (1) Obtain subcutaneous fat from a mammal, cut it into minced meat, add 1-3 mg / mL collagenase solution, transfer it to a tissue dissociation tube, dissociate it using a tissue dissociator, place it in a 37°C water bath, and digest it for 60-90 minutes, using a tissue dissociator to dissociate it once every 15-20 minutes; then add an equal volume of high-glucose DMEM containing 10% fetal bovine serum to terminate the digestion, and filter it with a 70-150 μm cell sieve, wherein the volume ratio of the minced meat to the collagenase solution is 1:5; (2) Centrifuge the digestion solution at 800-1500 rpm at room temperature for 5-10 min, discard the supernatant, and retain the cell pellet; (3) Add 3-5 times the volume of red blood cell lysis buffer to the cell pellet to resuspend it. Lyse it at room temperature for 5-10 min and then centrifuge it. Discard the supernatant and keep the cell pellet. (4) Resuspend the cell pellet in high-glucose DMEM containing 20% ​​FBS and inoculate it into a cell culture dish. After subculturing, use high-glucose DMEM containing 10% FBS as the complete culture medium.

3. The method for adipogenic differentiation of mammalian adipocytes according to claim 1, characterized in that: During adipogenic differentiation, the differentiation process was monitored by Oil Red O staining, BODIPY staining, TAG content determination and / or Western blot technology.

4. The method for adipogenic differentiation of mammalian adipocytes according to claim 3, characterized in that: During adipogenic differentiation, the differentiation process was monitored by BODIPY staining technique. The detection steps are as follows: (1) Discard the culture medium and rinse 1-2 times with PBS; (2) Add cell fixative, fix for 10-30 min, and rinse with PBS 2-4 times; (3) Add appropriate amount of 1-5 μmol / L BODIPY working solution and 1-5 μg / mL DAPI solution and stain for 10-15 min in the dark; (4) Discard the staining solution and rinse with PBS three times; (5) Observe and take photos using a fluorescence microscope.

5. The method for adipogenic differentiation of mammalian adipocytes according to claim 3, characterized in that: The Western blot technique uses ADIPOQ, FABP4, PPARγ and CEBPα as differentiation marker proteins, and uses TUBB and ACTB as internal reference parameters for monitoring.

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