CN117625527A - Method for separating, identifying and adipogenic differentiating mammalian fat precursor cells, composition and application - Google Patents

Method for separating, identifying and adipogenic differentiating mammalian fat precursor cells, composition and application Download PDF

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CN117625527A
CN117625527A CN202311756120.XA CN202311756120A CN117625527A CN 117625527 A CN117625527 A CN 117625527A CN 202311756120 A CN202311756120 A CN 202311756120A CN 117625527 A CN117625527 A CN 117625527A
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王守伟
梁小娟
李雨爽
李莹莹
唐铎
李石磊
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China Meat Research Centre
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Abstract

The invention belongs to the technical field of in-vitro differentiation of cells, and provides a method and a composition for separating, identifying and adipogenic differentiation of fat precursor cells of mammals and application thereof. The results show that the adipogenic differentiation method has good beneficial effects, is an ideal method for researching the differentiation of fat cells, and has important application value for the fields of animal husbandry, biomedicine and biological cultivation meat.

Description

Method for separating, identifying and adipogenic differentiating mammalian fat precursor cells, composition and application
Technical Field
The invention belongs to the technical field of in-vitro cell differentiation, and particularly relates to a method for separating, identifying and adipogenic differentiating mammalian fat precursor cells, a composition and application thereof.
Background
With the increasing interest in sustainable foods and animal welfare, biological breeding meats are receiving increasing attention as a potential alternative. The biological cultivated meat is also called cultivated meat, cell cultivated meat, clean meat and the like, and is a novel meat food which is formed by controlling the rapid proliferation, directional differentiation and collecting and processing of animal cells in an in-vitro culture mode. Wherein the development of animal adipose tissue and the differentiation of adipose cells are of great significance to the research of meat quality and diseases.
In the traditional animal husbandry, the quality and fat content of pork are generally affected by pig breeds and feeding conditions, and the development process of pig fat cells can be controlled and regulated more accurately by using a fat cell differentiation technology, so that fine regulation and control of the meat quality are realized, the consistency and quality of the meat quality are improved, and the market demands of different types of pork are met. For example, by promoting the differentiation of pig fat precursor cells, the fat content in pork can be increased, the meat quality can be improved, and the pork is more suitable for the taste demands of consumers, so that high-quality meat cells can be produced under in vitro conditions, and a foundation is provided for the production of biological cultured meat.
Methods have been used to promote differentiation of porcine adipocytes, such as medium compositions, gene regulation methods, and small molecule compounds. However, these methods have some limitations such as stability, inefficiency, side effects, and the like. For example, a common method of mammalian adipocyte differentiation is the Cocktail method (Cocktail), specifically a hormonal mixture of Insulin (INS), dexamethasone (DEX) and 3-isobutyl-1-methylxanthine (IBMX). However, the method has low differentiation efficiency, uneven differentiation and poor stability. Thus, there is a need for a more efficient and viable method to overcome these technical problems, thereby promoting differentiation and maturation of porcine fat precursor cells.
The specification of China invention CN202210418352.3 describes a preparation method of yak precursor fat cells, wherein the kidney week and subcutaneous fat of calf in one week of birth are subjected to primary culture in vitro by a type I collagenase digestion method, low-concentration pancreatin digestion and microscopic observation are combined with physical shock to perform subculture, and an induced differentiation culture medium is modified to perform induced differentiation culture, so that mature yak fat cells are obtained. The cell obtained by the patent has good morphology, fast cell proliferation and efficient induced differentiation treatment, is a more effective precursor fat cell culture method, and provides a necessary carrier for researching the molecular mechanism of yak body fat deposition.
The above methods have a certain effect, but the differentiation efficiency, differentiation uniformity and stability still need to be improved, and a separation and differentiation method with few side effects is also currently required.
Disclosure of Invention
In order to solve the technical problems, the invention provides a method for separating, identifying and adipogenic differentiating mammalian fat precursor cells, a composition and application thereof, and the method has the advantages of high differentiation efficiency, uniform differentiation, good stability and few side effects; the operation method is simple and efficient to realize the differentiation and maturation of the pig fat cells.
A method for the isolation, identification and adipogenic differentiation of mammalian adipose precursor cells solving the above technical problems, comprising the steps of:
step one, isolating mammalian fat precursor cells: using a mechanical method in combination with a collagenase digestion method;
step two, identifying the mammalian fat precursor cells: using cellular immunofluorescence;
step two, lipogenic induction for promoting in vitro differentiation of mammalian fat precursor cells: inducing adipogenic differentiation of the adipose precursor cells into mature adipocytes by a combination of a plurality of adipogenic induction factors;
step four, identifying mature adipocytes of the mammal: identification was performed using oil red O staining, BODIPY staining, TAG (triglyceride) assay and/or Western blot techniques.
The mammal is human and other mammals, preferably other mammals are pigs, cattle, horses, sheep, mice, etc.
In an optimized scheme, the step one of separating the mammalian fat precursor cells specifically comprises the following steps:
(1) Obtaining subcutaneous fat of mammals, cutting into minced meat, adding 1-3 mg/mL collagenase solution, transferring to a tissue dissociation tube, dissociating by using a tissue dissociation device, placing in a water bath kettle at 37 ℃, digesting for 60-90 min, and dissociating once every 15-20 min; adding an equal volume of high-sugar DMEM containing 10% fetal bovine serum, stopping digestion, and filtering with a cell sieve of 70-150 μm; preferably, filtration is performed with a 100 μm cell sieve; the volume and dosage ratio of the minced meat to the collagenase solution is 1:5, a step of; wherein the collagenase is type I collagenase, and the mass concentration of the type I collagenase solution is 2 mg/mL;
(2) Centrifuging the digestive juice at 800-1500 rpm at normal temperature for 5-10 min, discarding supernatant, and retaining cell precipitate; preferably, the digest is centrifuged at 1000 rpm for 8 minutes at ambient temperature;
(3) Adding 3-5 times of volume of red blood cell lysate of minced meat into the cell sediment for resuspension, performing room temperature pyrolysis for 5-10 min, centrifuging, discarding supernatant, and reserving the cell sediment; preferably, the ice bath is not performed at room temperature;
(4) Cells were resuspended in high-glucose DMEM containing 20% FBS and then inoculated into cell culture dishes, and 10% FBS-containing high-glucose DMEM was used as complete medium after passaging.
The induction period of the method is 8 days, lipid drops appear from the next day, and then the lipid drops gradually increase and increase.
In an optimized scheme, the step two comprises the step of identifying the fat precursor cells obtained in the step one by adopting an immunofluorescence technology; preferably, the fat precursor cells in step one are characterized by cellular immunofluorescence using PREF1 as primary antibody; further preferably, the method specifically comprises the following steps:
(1) Inoculating cells into 24-well cell culture plates on the first day;
(2) The next day, the culture medium is discarded, after PBS washing, 300-500 mu L of cell fixing solution is added for fixing for 10-30 min, the fixing solution is discarded, and PBS is used for washing for 2-4 times;
(3) Adding 0.1% -1% PBSTr for cell permeation for 15-20 min, discarding PBSTr, and washing with PBS for 2-4 times;
(4) Adding 300-500 mu L of 0.2% -2% BSA, and sealing for 45-60 min at room temperature;
(5) Removing the blocking solution, adding 100-200 μl of primary PREF1 antibody (rabbit source) (dilution ratio 1:50-1:100), standing at 4deg.C overnight, and washing with PBS for 2-4 times;
(6) Adding 100-200 mu L goat anti-rabbit secondary antibody (dilution ratio is 1:100-1:200), and incubating at room temperature for 1-2 h;
(7) PBS (phosphate buffer solution) is used for washing for 2-4 times, 100-200 mu L of 1-5 mu g/mL DAPI staining solution is added for cell nucleus staining;
(8) After washing 2-4 times with PBS, observation was performed by using a fluorescence microscope and photographing was performed.
The third step specifically comprises the following steps: specific small molecule compounds are added to the cell culture medium to directly promote maturation of adipocytes:
(1) Inoculating cells to a 6-hole plate for culture, and performing two-dimensional culture; or inoculating the cells into a three-dimensional culture roller bottle 10 5 cell/mL medium, 1 piece microcarrier/10 mL medium, rotation speed set at 25 rpm,10 min;0 rpm,50 min,24 cycles, and then the rotation speed is set to be 30 rpm, and three-dimensional culture is carried out;
(2) After the cells were full (confluence reached 100%), cultivation was continued for 2 d;
(3) Changing the complete culture medium into a lipid differentiation culture medium, and inducing differentiation culture 2 d;
(4) Changing to a maintenance differentiation culture medium, and maintaining differentiation culture 2 d;
(5) Changing to complete culture medium, continuing culturing for 4 d, and changing culture medium every 2 days;
wherein the components in the adipogenic differentiation medium comprise high glucose DMEM, insulin, glucocorticoid, IBMX, thiazolidinedione, sodium fatty acid and 2-10% fetal bovine serum, and the components in the differentiation medium comprise high glucose DMEM, insulin, sodium fatty acid, thiazolidinedione and 2-10% FBS; the complete medium is 10% FBS high glucose DMEM; preferably, the thiazolidinedione is one of troglitazone and rosiglitazone, and/or the fatty acid sodium is sodium oleate, and/or the glucocorticoid is one of dexamethasone or hydrocortisone.
The induction period of the method is 8 days, lipid drops appear from the next day, and then the lipid drops gradually increase and increase.
In a further optimized scheme, the method of the invention identifies the mature adipocytes differentiated in the step (four) through oil red O staining, BODIPY staining, TAG content measurement and/or Western blot technology; preferably, the differentiated adipocytes are collected, the total cell proteins are extracted, and the expression of differentiation marker proteins ADIPOQ, FABP4, PPARgamma and CEBP alpha is detected by a Western blot technique, and TUBB and ACTB are used as internal references at the same time; preferably, BODIPY staining is used, comprising the steps of:
(1) Discarding the culture solution, and rinsing with PBS for 1-2 times;
(2) Adding cell fixing solution, fixing for 10-30 min, and rinsing with PBS for 2-4 times;
(3) Adding 1-5 mu mol/L BODIPY working solution and 1-5 mu g/mL DAPI solution, and dyeing for 10-15 min in dark place;
(4) Discarding the staining solution, and rinsing with PBS for 3 times;
(5) Observed using a fluorescence microscope and photographed.
A composition for isolation, identification and adipogenic differentiation of mammalian adipose precursor cells according to the present invention comprises insulin, IBMX, glucocorticoid, sodium fatty acid and thiazolidinedione; preferably, the thiazolidinedione is one of troglitazone and Rosiglitazone (RSG), and/or the sodium fatty acid is sodium oleate, and/or the glucocorticoid is one of Dexamethasone (DEX) or hydrocortisone; or the composition is composed of insulin, IBMX, dexamethasone, sodium oleate, and rosiglitazone.
Insulin can promote glucose in blood circulation to enter hepatic cells, muscle cells, fat cells and other tissue cells to synthesize glycogen, so that blood sugar is reduced, and synthesis of fat and protein is promoted.
Glucocorticoids are extremely important regulating molecules in the organism, play an important role in regulating the development, growth, metabolism, immune function and the like of the organism, and are the most important regulating hormones for stress response of the organism; has antiinflammatory, antitoxic, antiallergic, antishock, and nonspecific immunity inhibiting effects.
IBMX is a Phosphodiesterase (PDE) inhibitor, also known as 3-isobutyl-1-methylxanthine, which has a vasodilating effect by increasing intracellular cAMP concentration and increasing calcium ion influx by inhibiting cAMP cleavage by inhibiting phosphodiesterase F-iii which cleaves cAMP, and by producing positive myogenic effects, in addition to positive myogenic effects, phosphodiesterase inhibitors have a vasodilating effect by increasing vascular smooth muscle intracellular cAMP content.
Rosiglitazone activates PPAR-gamma nuclear receptors and is involved in the regulation of fatty acid metabolism.
Troglitazone, also known as Razilin, regulates adipocyte differentiation and lipid metabolism.
Sodium fatty acid, sodium stearyl acid, is a compound consisting of two parts, hydrophobic and hydrophilic, and is used as an anionic surfactant. Is also a triglyceride synthesis feedstock.
The invention relates to a adipogenic differentiation medium for promoting pig fat precursor cell differentiation, which comprises basic medium high-sugar DMEM, wherein the basic medium high-sugar DMEM comprises 5-10 mug/mL of insulin, 0.2-0.8 mug/mL of dexamethasone, 0.3-0.6 mmol/L of IBMX, 1-5 mug/L of rosiglitazone, 100-200 mug/L of sodium oleate and 2-10% of fetal bovine serum by taking the volume of the basic medium high-sugar DMEM as a reference; preferably, the composition contains 5. Mu.g/mL of insulin, 0.4. Mu.g/mL of dexamethasone, 0.5mmol/L of IBMX, 1. Mu. Mol/L of thiazolidinedione, 150. Mu. Mol/L of sodium fatty acid and 2-10% of fetal bovine serum; preferably, the fetal bovine serum is 10%.
The invention relates to a maintenance differentiation culture medium for promoting pig fat precursor cell differentiation, which comprises a basal medium high-sugar DMEM, wherein the basal medium high-sugar DMEM comprises 5-10 mug/mL of insulin, 1-5 mug/L of rosiglitazone, 100-200 mug/L of sodium oleate and 2-10% fetal bovine serum by taking the volume of the basal medium high-sugar DMEM as a reference; preferably, insulin is 5. Mu.g/mL, thiazolidinedione is 1. Mu. Mol/L, sodium fatty acid is 150. Mu. Mol/L and 2-10% fetal bovine serum; preferably, the fetal bovine serum is 10%.
The application of the culture medium in the invention is that the adipogenic differentiation culture medium and the maintenance differentiation culture medium are applied, and the application comprises the following steps:
(1) Mammalian fat precursor cells reached 100% confluence and continued to be cultured 2 d;
(2) Discarding the old culture medium, and adding a adipogenic differentiation culture medium to induce differentiation 2 d;
(3) Removing the adipogenic differentiation medium, and adding a maintenance differentiation medium to maintain differentiation culture 2 d;
(4) The differentiation maintaining culture medium is discarded, a complete culture medium is added, and the culture is continued for 4 d, and the culture medium is replaced every 2 d;
(5) Observed using a microscope and photographed.
The application of the composition in the invention is the application in preparing medicines or compositions for preventing, relieving and/or treating lipodystrophy, medicines for regulating and controlling fat development and metabolism, and application in a adipogenic differentiation culture medium; wherein the effective components are insulin, glucocorticoid, IBMX, thiazolidinedione and sodium fatty acid; preferably, the thiazolidinedione is one of troglitazone and rosiglitazone, and/or the fatty acid sodium is sodium oleate, and/or the glucocorticoid is one of dexamethasone or hydrocortisone.
Further to the application in preparing a kit of adipogenic differentiation additive of fat cells and a kit for improving a lipodystrophy animal model.
Further, the application in preparing the biological cultivation meat.
The application of the adipogenic differentiation culture medium comprises basal medium high sugar DMEM, fetal bovine serum, insulin, dexamethasone, IBMX, rosiglitazone and sodium oleate.
The active ingredients of the prepared medicine are a composition of insulin, dexamethasone, IBMX, rosiglitazone and sodium oleate.
The method, the composition and the application of the invention can realize the differentiation and the maturation of the mammalian fat cells under simpler and more efficient conditions; the differentiation efficiency is high, the differentiation is uniform, the stability is good, and the side effect is less; the method has a very good differentiation effect in a common culture mode such as two-dimensional culture or three-dimensional culture; can provide better tools and methods for animal husbandry, biomedical research and biological cultivation meat research, further understand the development process of the mammalian fat cells, and can be used for regulating and controlling fat development and metabolism and physiological and pathological processes related to the fat development and metabolism.
Drawings
FIG. 1 is a cell morphology of the isolated porcine fat precursor cells of example 5 of the present invention;
FIG. 2 is a graph showing the result of immunofluorescence assay of PREF1 from porcine fat precursor cells in example 5 of the present invention (note: blue is nucleus, red is PREF1 protein);
FIG. 3 is a graph showing the identification of mature adipocytes in swine of example 7 of the present invention;
fig. 4 is a graph of the measurement results of triglyceride content of differentiated 0 d and differentiated 8 d porcine adipocytes in example 7 of the present invention (p <0.001 indicates that the difference is extremely remarkable);
FIG. 5 is a graph showing the results of detection of expression of adipogenic differentiation-related genes in example 7 of the present invention;
FIG. 6 is a graph showing the results of staining lipid-forming differentiated lipid droplets in three-dimensional culture of porcine fat precursor cells in example 8 of the present invention (Scale bar:100 μm);
FIGS. 7 and 8 are graphs comparing differentiation results of 8 different adipogenic differentiation compositions of example 6 of the present invention.
Detailed Description
The invention is further illustrated by the following description of specific embodiments:
example 1 compositions for mammalian adipose precursor cell isolation, identification and adipogenic differentiation comprising insulin, IBMX, glucocorticoid, sodium fatty acid and thiazolidinedione;
in this embodiment, the thiazolidinedione is one of troglitazone and Rosiglitazone (RSG), and/or the fatty acid sodium is sodium oleate, and/or the glucocorticoid is one of Dexamethasone (DEX) or hydrocortisone; or the composition is composed of insulin, IBMX, glucocorticoid, sodium oleate and rosiglitazone.
Further, the culture medium for promoting the differentiation of the fat precursor cells comprises the following components in parts by weight: the basal medium, high glucose DMEM, contains 5 μg/mL insulin, 0.4 μg/mL dexamethasone, 0.5mmol/L IBMX, 1 μmol/L rosiglitazone, 150 μmol/L sodium oleate and 2% fetal bovine serum.
The maintenance differentiation culture medium for promoting pig fat precursor cell differentiation comprises the following components in parts by weight: the basal medium, high sugar DMEM, contains 5 μg/mL insulin, 1 μmol/L rosiglitazone, 150 μmol/L sodium oleate and 2% fetal bovine serum based on the basal medium.
Example 2 a medium for the isolation, identification and adipogenic differentiation of mammalian adipose precursor cells, wherein the medium promoting the differentiation of adipose precursor cells consists of the following components and amounts: the basal medium high-sugar DMEM contains 5 mu g/mL of insulin, 0.4 mu g/mL of dexamethasone, 0.5mmol/L of IBMX, 1 mu mol/L of rosiglitazone, 150 mu mol/L of sodium oleate and 10% of fetal bovine serum by volume of the basal medium.
The maintenance differentiation culture medium for promoting pig fat precursor cell differentiation comprises the following components in parts by weight: the basal medium high-sugar DMEM contains 5 mu g/mL of insulin, 1 mu mol/L of rosiglitazone, 150 mu mol/L of sodium oleate and 10% of fetal bovine serum based on the volume of the basal medium.
Example 3 adipogenic differentiation Medium for promoting differentiation of porcine fat precursor cells wherein the Medium comprises basal medium high glucose DMEM containing 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 based on basal medium DMEM volume.
A maintenance differentiation medium for promoting pig fat precursor cell differentiation, the medium comprising basal medium high sugar DMEM containing 5 μg/mL insulin, 1 μmol/L rosiglitazone, 100 μmol/L sodium oleate and 8% fetal bovine serum based on basal medium high sugar DMEM volume.
Example 4 adipogenic differentiation Medium for promoting differentiation of porcine fat precursor cells comprising basal medium high glucose DMEM containing insulin 10 μg/mL, glucocorticoid 0.8 μg/mL, IBMX0.6 mmol/L, rosiglitazone 5 μmol/L, sodium oleate 200 μmol/L and 5% fetal bovine serum based on basal medium high glucose DMEM volume.
A maintenance differentiation medium for promoting pig fat precursor cell differentiation, the medium comprising basal medium high sugar DMEM containing insulin 10 μg/mL, rosiglitazone 5 μmol/L, sodium oleate 200 μmol/L and 5% fetal bovine serum based on basal medium high sugar DMEM volume.
Example 5 methods for isolation, identification and differentiation of porcine fat precursor cells.
In this example, porcine adipose tissue was minced to 1 mm using an ophthalmic scissors 3 Then adding type I collagenase for digestion, then sieving cells, and centrifuging to obtain the pig fat precursor cells. The fat precursor cells of pigs were found to be irregularly long fusiform, triangular, etc. by microscopic observation (fig. 1). The obtained pig fat precursor cells are identified by adopting a cell immunofluorescence method, and the result shows that almost all cells express PREF1 protein (marker protein of fat precursor cells), which indicates that the pig fat precursor cells are successfully separated (figure 2), and materials are provided for researching the in vitro differentiation of the pig fat precursor cells; the specific method comprises the following steps:
the isolation procedure for pig fat precursor cells was as follows:
(1) Killing piglets after anesthesia;
(2) Transferring the piglet bodies into a biosafety cabinet subjected to ultraviolet sterilization treatment, and wiping the whole body with 75% alcohol for sterilization;
(3) Separating the neck subcutaneous fat using a sterile scalpel, an ophthalmic scissors and an ophthalmic forceps;
(4) Subcutaneous fat was rinsed in 75% alcohol and then transferred to PBS solution;
(5) Removing macroscopic blood vessels, muscle and other impurities to obtain pig subcutaneous fat, and shearing the subcutaneous fat into pieces of about 1 mm 3 Ground meat emulsion of size;
(6) Adding 5 times volume of 2 mg/mL type I collagenase solution into a centrifuge tube containing minced meat; transferring to a dissociation tube;
(7) The dissociation tube is dissociated by a tissue dissociator and then is placed in a water bath kettle at 37 ℃ for digestion for 60 min, and the dissociation tube is dissociated once every 15 min;
(8) The digestion was stopped by adding an equal volume of complete medium (high-sugar DMEM with 10% fetal bovine serum) and filtered through a 100 μm cell sieve;
(9) Centrifuging the digestive juice at a speed of 1,000 rpm for 8 min at normal temperature; discarding the supernatant, and retaining the cell pellet;
(10) Adding 3 times volume of red blood cell lysate (ACK Lysis Buffer) into the cell sediment for resuspension, performing room temperature pyrolysis for 10 min, centrifuging, discarding the supernatant, and reserving the cell sediment; standing at room temperature without ice bath;
(11) Cells were resuspended in high-carbohydrate DMEM containing 20% FBS, counted and plated at appropriate cell densities on 10 cm cell culture dishes, and after passaging, 10% FBS in high-carbohydrate DMEM was used as complete medium.
Identifying the fat precursor cells obtained in the step one by adopting immunofluorescence technology; immunofluorescence of cells using PREF1 as primary antibody to characterize the fat precursor cells in step one, immunofluorescence of porcine fat precursor cells was identified as follows:
(1) Inoculating cells into 24-well cell culture plates on the first day;
(2) The next day, the medium was discarded and washed 3 times with PBS, 300. Mu.L of cell fixative was added to the wells and the wells were fixed for 15 min. Discarding the fixing solution, washing with PBS for 3 times, each time for 5 min;
(3) Adding 0.1 PBSTr for cell permeation for 15 min, discarding the PBSTr, and washing with PBS for 3 times each for 5 min;
(4) 300. Mu.L of 0.2% BSA was added and blocked at room temperature for 60 min;
(5) Removing the blocking solution, adding 200 mu L of diluted PREF1 primary antibody (rabbit source) in a dilution ratio of 1:50-1:100, and standing at 4 ℃ overnight; washing with PBS for 3 times, each time for 5 min;
(6) 200. Mu.L of diluted goat anti-rabbit secondary antibody (dilution ratio 1:100-1:200) is added, and the mixture is incubated at room temperature for 1 h;
(7) Washing with PBS for 5 min for 3 times, diluting 100. Mu.L of 2. Mu.g/mL DAPI to 1. Mu.g/mL with PBS, and adding 200. Mu.L of staining solution into the wells for nuclear staining;
(8) PBS was washed 3 times for 5 min each, after which it was observed with a fluorescence microscope and photographed.
Example 6 screening of methods for promoting adipogenic differentiation of fat precursor cells
The steps of adipogenic induced differentiation of pig fat precursor cells are as follows;
(1) Inoculating cells into a 6-well plate for two-dimensional culture;
(2) The confluence of the pig fat precursor cells reaches 100 percent, and the pig fat precursor cells are continuously cultured for 2 d;
(3) The original complete culture medium is changed into a lipid differentiation culture medium, and induced differentiation culture is carried out on 2 d;
(4) Changing to a maintenance differentiation culture medium, and maintaining differentiation culture 2 d;
(5) Changing to complete culture medium, continuing culturing for 4 d, and changing culture medium every 2 days;
(6) The images were observed and photographed using a general microscope.
Wherein the components in the adipogenic differentiation medium are high-sugar DMEM, insulin, dexamethasone, IBMX, rosiglitazone, sodium oleate and 2% or 10% fetal bovine serum, and the components in the maintenance differentiation medium are high-sugar DMEM, insulin, sodium oleate, rosiglitazone and 2% or 10% FBS; complete medium was 10% FBS in high-sugar DMEM.
In this example, under otherwise identical conditions, 4 adipocyte inducer combinations and 2 serum concentrations were compared, each: (1) Cocktail (Cocktail method), sodium oleate and 2% FBS; (2) Cocktail, sodium oleate, RSG (rosiglitazone) and 2% fbs; (3) Cocktail and 2% FBS; (4) 2% FBS of Cocktail, RSG; (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.
As shown in FIGS. 7 and 8, the effect of inducing differentiation by serum at a high concentration was good in the case of the same inducer, whereas the effect of cell differentiation was good in the case of using the cocktail method in combination with sodium oleate and rosiglitazone at the same concentration. Namely, the "Cocktail, sodium oleate, 10% FBS of RSG" group was most effective in differentiation.
The adipogenic differentiation medium is: high sugar dmem+cocktail+sodium oleate+rsg+10% fbs; the maintenance differentiation medium was: high sugar dmem+ins (insulin) +sodium oleate+rsg+10% fbs. Wherein, insulin in the adipogenic differentiation medium is 5 mug/mL, dexamethasone is 0.4 mug/mL, IBMX is 0.5mmol/L, rosiglitazone is 1 mug/L, sodium oleate is 150 mug/L; insulin 5. Mu.g/mL, rosiglitazone 1. Mu. Mol/L, sodium oleate 150. Mu. Mol/L in the differentiation medium was maintained.
EXAMPLE 7 identification of mature adipocytes and expression of differentiation-related Gene
The method of staining adipocytes generally includes oil red O staining, BODIPY staining, and the like. In addition, the observation can be directly performed without dyeing.
Oil red O is a fat-soluble dye that is highly soluble in fat and specifically colors neutral fats such as intracellular triglycerides. BODIPY is a near infrared short wavelength fluorescent dye that acts specifically on oil droplets composed of neutral lipids.
In the present invention, direct observation and staining of undifferentiated (0 d) and differentiated 8 d (10% FBS composition using Cocktail, sodium oleate, RSG) cells were performed using the direct observation method and three methods of oil red O staining and BODIPY staining.
As a result, as shown in FIG. 3, vacuolated lipid droplets were observed in cells differentiated by 8 d, and after staining, these lipid droplets were stained with oil red O to bright red and BODIPY to green, indicating that the adipogenic differentiation method using the 10% FBS composition of "Cocktail, sodium oleate, RSG successfully promoted adipogenic differentiation of pig fat precursor cells into mature adipocytes.
The core component of the lipid droplets is triglyceride, the invention collects the fat cells differentiated by 0 d and 8 d, and the triglyceride content is determined by using a cell triglyceride determination kit.
The results are shown in figure 4, where the content of Triglycerides (TAGs) in differentiated 8 d cells is significantly increased compared to undifferentiated fat precursor cells (0 d). The specific detection method is shown in a kit (product number E1013) for measuring the Triglyceride (TG) content of the Prilet tissue cells by an enzyme method.
According to the invention, adipocytes differentiated by 0 d, 2 d, 4 d, 6 d and 8 d are collected, total cell proteins are extracted, expression of PREF1, ADIPOQ, FABP4, PPARgamma and CEBP alpha is detected by a Western blot technology, and TUBB and ACTB are used as internal references at the same time.
As a result, as shown in FIG. 5, the expression level of precursor adipocyte DLK1 was gradually decreased from 2 d, and the expression level was very weak in the cells differentiated by 8 d, which was consistent with the expected result. Whereas the expression of ADIPOQ, FABP4, pparγ, cebpα gradually increased with the differentiation expression amount, FABP4 peaked as a marker of terminal differentiation at day 8 of differentiation, as shown in fig. 4. The detection method comprises the following steps:
mature adipocyte oil red O staining was used as follows:
(1) Discarding the culture solution, and adding PBS to gently rinse for 1 time;
(2) Adding cell fixing solution, fixing for 10 min, and rinsing with PBS for 2 times;
(3) Adding a proper amount of staining washing liquid to cover the cells 20 and s;
(4) Discarding the dyeing washing liquid, adding a proper amount of modified oil red O dyeing liquid, and dyeing for 15 min in a dark place;
(5) Discarding the modified oil red O staining solution, adding a proper amount of staining washing solution, and standing for 30 s;
(6) The staining wash was discarded, washed with PBS 20 s, observed under a mirror and photographed.
Dyeing with BODIPY dye liquor, wherein the steps are as follows:
(1) The culture broth was discarded and rinsed 1 time with PBS;
(2) Adding cell fixing solution, fixing for 10 min, and rinsing with PBS for 3 times;
(3) Adding 5 mu mol/L BODIPY working solution and 5 mu g/mL DAPI solution, and dyeing for 10 min in dark;
(4) Discarding the staining solution, rinsing with PBS for 3 times, each time for 5 min;
(5) Observed under a fluorescence microscope and photographed.
Cell protein extraction:
(1) Discarding the culture solution, and adding PBS for rinsing for 1 time;
(2) Adding appropriate amount of cell lysate (300 μl per well of 6-well plate) containing protease inhibitor, and standing on ice for 10 min;
(3) Scraping the cells from the bottom of the hole from left to right by scraping the cells from top to bottom, and blowing and uniformly mixing the cells by using a pipetting gun;
(4) The cell lysate was collected in a 1.5 mL centrifuge tube and centrifuged at 12,000 rpm at 4℃for 30 min;
(5) Transferring the supernatant to a new 1.5 mL centrifuge tube, placing the supernatant, namely the protein, at-80 ℃ for later use.
Western blot：
(1) Taking out the protein in a refrigerator at-80deg.C, thawing on ice, mixing 20 μL of protein with 5 μL of 5×protein loading buffer solution, boiling at 100deg.C for 10 min to denature the protein;
(2) Preparing 10% -12% SDS-PAGE gel;
(3) 10 mu L of denatured protein is added to each lane, and 5 mu L of pre-dyed protein Marker is reserved in one lane;
(4) Setting 120V electrophoresis conditions, and performing electrophoresis for 15 min and 200V electrophoresis for 40 min until bromophenol blue indicator strips migrate to about 1 cm from the lower end of gel, and stopping electrophoresis;
(5) Setting constant current 400 and mA by using a wet transfer method, and transferring for about 30 minutes to a PVDF film;
(6) Placing the PVDF membrane into an antibody incubation box containing a sealing solution, wherein the room temperature is 1 h;
(7) The sealing liquid is used for sealing according to the proportion of 1: diluting the primary antibody at 5000, adding the primary antibody into an antibody incubation box, and incubating overnight at 4 ℃;
(8) TBST washes the membrane for 3 times, each time for 10 min;
(9) The following is 1 with TBST: diluting the secondary antibody at 5000, adding the secondary antibody into an antibody incubation box, and incubating at room temperature for 1 h;
(10) TBST washes the membrane for 3 times, each time for 10 min;
(11) Detection was performed with ECL chemiluminescent solution and observations and photographs were taken using a chemiluminescent imaging system.
EXAMPLE 8 application of pig fat precursor cell adipogenic differentiation in three-dimensional culture
The three-dimensional culture of cells is more similar to the physiological environment in vivo than the traditional two-dimensional culture. The lipid-forming differentiation medium comprising "high sugar DMEM, cocktail, sodium oleate, RSG and 10% fbs" was added to the three-dimensional culture roller bottle, and after 2 days, the differentiation method of changing the composition comprising "high sugar DMEM, INS, sodium oleate, RSG and 10% fbs" maintenance differentiation medium was applied to the three-dimensional culture. Upon BODIPY staining, it was found that adipogenic differentiation of fat precursor cells into mature adipocytes was successful also in the case of three-dimensional culture (fig. 6). The pig fat precursor cell differentiation method is applicable to two-dimensional and three-dimensional culture.
The specific method for three-dimensionally culturing the pig fat precursor cells into lipid differentiation is as follows:
(1) Inoculating cells into a roller bottle, 10 5 cell/mL medium, 1 piece microcarrier/10 mL medium, rotation speed set at 25 rpm,10 min;0 rpm,50 min,24 cycles, followed by a rotational speed set to 30 rpm;
(2) After the cells are full, culturing is continued for 2 d;
(3) Changing the complete culture medium into a lipid differentiation culture medium, and inducing differentiation culture 2 d;
(4) Changing to a maintenance differentiation culture medium, and maintaining differentiation culture 2 d;
(5) Changing to complete culture medium, continuing culturing for 4 d, and changing culture medium every 2 days;
the application of the culture medium is characterized in that the adipogenic differentiation culture medium and the maintenance differentiation culture medium are applied, and the steps are as follows:
(1) The confluence of animal fat precursor cells reaches 100 percent, and the culture is continued for 2 d;
(2) Discarding the old culture medium, and adding a adipogenic differentiation culture medium to induce differentiation 2 d;
(3) Removing the adipogenic differentiation medium, and adding a maintenance differentiation medium to maintain differentiation culture 2 d;
(4) The differentiation maintaining culture medium is discarded, a complete culture medium is added, and the culture is continued for 4 d, and the culture medium is replaced every 2 d;
(5) Observed using a microscope and photographed.
Fat drop BODIPY staining
(1) Sucking 1 mL cell suspension, settling the cells together with the microcarriers, discarding the supernatant medium, and rinsing once with PBS;
(2) Adding cell fixing solution, fixing for 10 min, and rinsing with PBS for 2 times;
(3) Adding proper amount of 5 mu mol/L BODIPY working solution and DAPI (simultaneously dying cell nuclei) to cover cells and dyeing for 10 min in dark;
(4) Discarding the staining solution, washing with PBS for 3 times, each time for 5 min;
(5) The photographs were scanned using a high content cell imaging system.
Example 9 otherwise as in example 5, wherein: in the step of separating pig fat precursor cells:
(6) Adding 5 times volume of type I collagenase solution of 1 or 3 mg/mL into a centrifuge tube containing minced meat, and transferring to a tissue dissociation tube;
(7) The tissue dissociation tube is dissociated by a tissue dissociation device and then is placed in a water bath kettle at 37 ℃ for digestion for 60 or 90 min, and the tissue dissociation device is used for dissociation once every 20 min;
(8) The digestion was stopped by adding an equal volume of complete medium (high-sugar DMEM with 10% fetal bovine serum) and filtered through a 70 or 150 μm cell sieve;
(9) Centrifuging the digestive juice at 800 or 1500 rpm for 5 or 10 min at normal temperature; discarding the supernatant, and retaining the cell pellet;
(10) Adding red blood cell lysate (ACK Lysis Buffer) with the volume of 5 times of that of the minced meat into the cell sediment for resuspension, performing centrifugation after 5 or 8 min of room temperature pyrolysis, discarding the supernatant, reserving the cell sediment, and standing at room temperature without ice bath;
(11) Cells were resuspended in high-carbohydrate DMEM containing 20% FBS, counted and plated at appropriate cell densities on 10 cm cell culture dishes, and passaged using 10% FBS-containing high-carbohydrate DMEM as complete medium.
The immunofluorescence identification steps of the pig fat precursor cells are as follows:
(1) Inoculating cells into 24-well cell culture plates on the first day;
(2) The next day, the medium was discarded, washed 3 times with PBS, 400 or 500. Mu.L of cell fixative was added to the wells, and fixation was performed for 15 min; discarding the fixing solution, washing with PBS for 2 or 4 times, each for 5 min;
(3) Adding 0.1-1% PBSTr for cell permeation for 20 min, discarding PBSTr, and washing with PBS for 2 or 4 times for 5 min each time;
(4) Adding 400 or 500 mu L of 0.2-2% BSA, and blocking at room temperature for 45 or 50 min;
(5) Discarding the blocking solution, adding 100-200 mu L of diluted PREF1 primary antibody (rabbit source) in the dilution ratio of 1:50-1:100, and standing at 4 ℃ overnight; PBS is washed for 2 or 4 times, each time for 5 min;
(6) Adding 100-200 mu L of diluted goat anti-rabbit secondary antibody (dilution ratio is 1:1000-1:200), and incubating at room temperature for 2 h;
(7) Washing with PBS for 2 or 4 times, each for 5 min, diluting 150 or 200 mu L of 1 or 5 mu g/mL DAPI to 1 mu g/mL with PBS, and adding 200 mu L of staining solution into the hole for cell nucleus staining;
(8) PBS was washed 2 or 4 times, 5 min each, after which it was observed with a fluorescence microscope and photographed.
The above does not mention the specific name of the medium, the complete medium, and the detection device used is a conventional device or instrument.
The above examples/experiments are only examples for clarity of illustration and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the invention.

Claims (10)

1. A method for the isolation, identification and adipogenic differentiation of mammalian adipose precursor cells, characterized in that: the method comprises the following steps:
step one, isolating mammalian fat precursor cells: using a mechanical method in combination with a collagenase digestion method;
step two, identifying the mammalian fat precursor cells: using cellular immunofluorescence;
step three, promoting in vitro induced differentiation of the mammalian fat precursor cells: differentiating fat precursor cells into mature fat cells by a combination of multiple adipogenic inducers;
step four, identifying mature adipocytes of the mammal: identification was performed using oil red O staining, BODIPY staining, TAG assay and/or Western blot techniques.
2. A method for the isolation, identification and adipogenic differentiation of mammalian adipose precursor cells according to claim 1, wherein: the step one of separating the pig fat precursor cells specifically comprises the following steps:
(1) Obtaining subcutaneous fat of mammals, cutting into minced meat, adding 1-3 mg/mL collagenase solution, transferring to a tissue dissociation tube, dissociating by using a tissue dissociation device, placing in a water bath kettle at 37 ℃, digesting for 60-90 min, and dissociating once every 15-20 min; adding an equal volume of high-sugar DMEM containing 10% fetal bovine serum, stopping digestion, and filtering with a cell sieve of 70-150 μm; preferably, filtration is performed with a 100 μm cell sieve; the volume and dosage ratio of the minced meat to the collagenase solution is 1:5, a step of;
(2) Centrifuging the digestive juice at 800-1500 rpm at normal temperature for 5-10 min, discarding supernatant, and retaining cell precipitate; preferably, the digest is centrifuged at 1000 rpm for 8 minutes at ambient temperature;
(3) Adding 3-5 times of volume of red blood cell lysate of minced meat into the cell sediment for resuspension, performing room temperature pyrolysis for 5-10 min, centrifuging, discarding supernatant, and reserving the cell sediment; preferably, the ice bath is not performed at room temperature;
(4) Cell pellets were resuspended in high-carbohydrate DMEM containing 20% FBS and inoculated into cell culture dishes, and after passaging, high-carbohydrate DMEM containing 10% FBS was used as complete medium.
3. A method for the isolation, identification and adipogenic differentiation of mammalian adipose precursor cells according to claim 1, wherein: the second step is to identify the fat precursor cells obtained in the first step by adopting immunofluorescence technology; preferably, the fat precursor cells in step one are characterized by cellular immunofluorescence using PREF1 as primary antibody; further preferably, the method specifically comprises the following steps:
(1) Inoculating cells into 24-well cell culture plates on the first day;
(2) The next day, the culture medium is discarded, after PBS washing, 300-500 mu L of cell fixing solution is added for fixing for 10-30 min, the fixing solution is discarded, and PBS is used for washing for 2-4 times;
(3) Adding 0.1% -1% PBSTr for cell permeation for 15-20 min, discarding PBSTr, and washing with PBS for 2-4 times;
(4) Adding 300-500 mu L of 0.2% -2% BSA, and sealing for 45-60 min at room temperature;
(5) Discarding the blocking solution, adding 100-200 mu L of PREF1 primary antibody, standing at 4 ℃ for incubation overnight, and washing with PBS for 2-4 times; preferably, the dilution ratio of the primary antibody is 1:50-1:100;
(6) Adding 100-200 mu L goat anti-rabbit secondary antibody, and incubating at room temperature for 1-2 h; preferably, the dilution ratio of the secondary antibody is 1:100-1:200;
(7) PBS (phosphate buffer solution) is used for washing for 2-4 times, 100-200 mu L of 1-5 mu g/mL DAPI staining solution is added for cell nucleus staining;
(8) After washing 2-4 times with PBS, observation was performed by using a fluorescence microscope and photographing was performed.
4. A method for the isolation, identification and adipogenic differentiation of animal fat precursor cells according to claim 1, characterized in that: the third step specifically comprises the following steps:
(1) Inoculating cells into a 6-well plate for two-dimensional culture; or inoculating the cells into a roller bottle 10 5 cell/mL medium, 1 piece microcarrier/10 mL medium, rotation speed set at 25 rpm,10 min;0 rpm,50 min,24 cycles, and then the rotation speed is set to be 30 rpm, and three-dimensional culture is carried out;
(2) After the cells are full, culturing is continued for 2 d;
(3) Changing the culture medium into a lipid differentiation culture medium, and inducing differentiation culture 2 d;
(4) Changing to a maintenance differentiation culture medium, and maintaining differentiation culture 2 d;
(5) Changing to complete culture medium, continuously culturing for 4 d, and changing culture solution every 2 days;
wherein the components in the adipogenic differentiation medium comprise high glucose DMEM, insulin, glucocorticoid, IBMX, thiazolidinedione reagent, fatty acid sodium and 2-10% fetal bovine serum, and the components in the differentiation medium comprise high glucose DMEM, insulin, fatty acid sodium, thiazolidinedione reagent and 2-10% FBS; preferably, the thiazolidinedione is one of troglitazone and rosiglitazone, and/or the fatty acid sodium is sodium oleate, and/or the glucocorticoid is one of dexamethasone or hydrocortisone.
5. A method for the isolation, identification and adipogenic differentiation of mammalian adipose precursor cells according to claim 1, wherein: identifying the mature adipocytes differentiated in the fourth step through oil red O staining, BODIPY staining, TAG content measurement and/or Western blot technology; preferably, the differentiated adipocytes are collected, the total cell proteins are extracted, and the expression of differentiation marker proteins ADIPOQ, FABP4, PPARgamma and CEBP alpha is detected by a Western blot technique, and TUBB and ACTB are used as internal references at the same time; preferably, the dyeing with BODIPY comprises the following steps:
(1) Discarding the culture solution, and rinsing with PBS for 1-2 times;
(2) Adding cell fixing solution, fixing for 10-30 min, and rinsing with PBS for 2-4 times;
(3) Adding proper 1-5 mu mol/L BODIPY working solution and 1-5 mu g/mL DAPI solution to dye for 10-15 min in dark place;
(4) Discarding the staining solution, and rinsing with PBS for 3 times;
(5) Observed using a fluorescence microscope and photographed.
6. A composition for the isolation, identification and adipogenic differentiation of mammalian adipose precursor cells, characterized in that: the composition comprises insulin, IBMX, a glucocorticoid, sodium fatty acid and a thiazolidinedione; preferably, the thiazolidinedione is one of troglitazone and rosiglitazone, and/or the fatty acid sodium is sodium oleate, and/or the glucocorticoid is one of dexamethasone or hydrocortisone; or the composition is composed of insulin, IBMX, dexamethasone, sodium oleate, and rosiglitazone.
7. A adipogenic differentiation medium for promoting differentiation of mammalian adipose precursor cells, characterized in that: the culture medium comprises basic culture medium high glucose DMEM, insulin 5-10 mu g/mL, glucocorticoid 0.2-0.8 mu g/mL, IBMX0.3-0.6 mmol/L, rosiglitazone 1-5 mu mol/L, sodium oleate 100-200 mu mol/L and 2-10% fetal bovine serum; preferably, insulin is 5. Mu.g/mL, glucocorticoid is 0.4. Mu.g/mL, IBMX is 0.5mmol/L, thiazolidinedione is 1. Mu. Mol/L, sodium fatty acid is 150. Mu. Mol/L and 2-10% fetal bovine serum; further preferably, the fetal bovine serum is 10%.
8. A maintenance differentiation medium for promoting differentiation of mammalian adipose precursor cells, characterized in that: the culture medium comprises a basal medium high-sugar DMEM, and contains 5-10 mu g/mL of insulin, 1-5 mu mol/L of rosiglitazone, 100-200 mu mol/L of sodium oleate and 2-10% of fetal bovine serum; preferably, insulin is 5. Mu.g/mL, thiazolidinedione is 1. Mu. Mol/L, sodium fatty acid is 150. Mu. Mol/L and 2-10% fetal bovine serum; further preferably, the fetal bovine serum is 10%.
9. Use of the medium according to claim 7 or 8, characterized in that: the adipogenic differentiation medium and the maintenance differentiation medium are applied, comprising the steps of:
(1) Mammalian fat precursor cells reached 100% confluence and continued to be cultured 2 d;
(2) Discarding the old culture medium, and adding a adipogenic differentiation culture medium to induce differentiation 2 d;
(3) Removing the adipogenic differentiation medium, and adding a maintenance differentiation medium to maintain differentiation culture 2 d;
(4) The differentiation maintaining culture medium is discarded, a complete culture medium is added, and the culture is continued for 4 d, and the culture medium is replaced every 2 d;
(5) The images were observed and photographed using a general microscope.
10. The composition as recited in claim 6, wherein: use of insulin, glucocorticoid, IBMX, thiazolidinedione and sodium fatty acid for the preparation of a medicament or composition for the prevention, alleviation and/or treatment of lipodystrophy, medicament for the regulation of fat development and metabolism; preferably, the thiazolidinedione is one of troglitazone and rosiglitazone, and/or the fatty acid sodium is sodium oleate, and/or the glucocorticoid is one of dexamethasone or hydrocortisone.
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