CN116410922A

CN116410922A - Method for separating and differentiating animal fat source mesenchymal precursor cells

Info

Publication number: CN116410922A
Application number: CN202310312197.1A
Authority: CN
Inventors: 余路阳; 蒋苑青
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2023-03-28
Filing date: 2023-03-28
Publication date: 2023-07-11

Abstract

The invention discloses a method for separating and differentiating mesenchymal precursor cells from animal fat, which is characterized by comprising the following steps: firstly, primarily separating animal fat to obtain a mixed cell population containing mesenchymal precursor cells, and then separating and purifying the mixed cell population by using a flow separation marker to obtain mesenchymal precursor cells from animal fat; and (3) culturing the mesenchymal precursor cells from animal fat, then performing contact inhibition, then adding an induced adipogenic differentiation medium for culturing, observing the cell morphology under a microscope, and when the cell morphology is observed to be normal and tiny lipid droplets appear and are positioned in cytoplasm of the cells, adding a maintenance differentiation medium for culturing, and performing identification and detection after differentiation is completed, so as to finish differentiation. The invention greatly improves the differentiation rate of the primary cells of the mice for inducing the adipogenesis, so that the primary cells of the mice are differentiated into mature adipocytes which are most in line with the anatomical in-situ property of the tissues and meet the clinical scientific research requirements.

Description

Method for separating and differentiating animal fat source mesenchymal precursor cells

Technical Field

The invention relates to the technical field of cell biology, in particular to a method for separating and differentiating animal fat-derived mesenchymal precursor cells.

Background

Periaortic adipose tissue (PVAT) is a fat depot attached to the aortic vessel wall, since special anatomical sites may not only have a vascular supporting function, but also act on other surrounding adipocytes and vessel walls through autocrine and paracrine actions, thereby affecting vascular homeostasis. PVAT plays a key role in the development and progression of cardiovascular disease. In vascular pathology, the volume of adipose tissue around the aortic blood vessels increases and gradually becomes dysfunctional, and its cellular composition and molecular characteristics also change. PVAT dysfunction is mainly characterized by its inflammatory character, oxidative stress, reduced vascular protective adipocyte-derived relaxants, and increased paracrine factors such as antibiotic, leptin, cytokines TNF- α and chemokines such as MCP-1, etc. These adipocyte-derived factors stimulate and coordinate inflammatory cell infiltration, which mainly includes T cells, macrophages, dendritic cells, B cells, and NK cells. Also, protective factors such as adiponectin secreted by periaortic fat can reduce the production of NADPH oxidase superoxide and increase the bioavailability of nitric oxide in the vessel wall, but pro-inflammatory factors secreted by periaortic fat induce dysfunction of vascular oxidase and nitric oxide synthase eNOS in endothelium, vascular smooth muscle cells and adventitia fibroblasts if they are in an inflammatory environment. All of these events relate dysfunctional perivascular aortic fat to vascular dysfunction, and these mechanisms are important in many cardiovascular diseases including atherosclerosis, hypertension, diabetes and obesity, and increasing researchers are interested in perivascular aortic adipose tissue studies.

Since 80% -90% of the mature adipocytes in cytoplasm are composed of lipid droplets, and the cells cannot be subjected to adherent subculture after separation and purification of terminally differentiated cells, the method brings great difficulty to related research of perivascular adipose tissues. The adipose-derived precursor mesenchymal cells are stem cells with multi-differentiation potential, which are obtained by separating and purifying adipose tissues, can be cultured and amplified in vitro on a large scale, have stable properties, differentiate into cells of cartilage, bone formation, fat, nerve and the like under the action of certain induction factors, and are widely applied to the fields of clinical research, medical science and the like. The primary adipose-derived precursor mesenchymal stem cells are more favored in scientific research because they are capable of reducing the physiological functions, related properties and intrinsic regulatory mechanisms of the adipose tissue in the body than the common adipose precursor stem cell lines. The mice are used as the optimal model organism for researching atherosclerosis, the gene modification and breeding are easy, and a large amount of adipose tissue around the aortic blood vessels can be easily obtained, so that the precursor mesenchymal cells derived from the adipose tissue around the aortic blood vessels of the mice are used as the research object, and the primary cell separation culture and differentiation scheme is continuously practiced.

In the existing manufacturing process, in vitro separation, culture and differentiation methods for primary precursor mesenchymal cells derived from adipose tissue around the blood vessels of the aorta of mice are very lacking. The technical problems to be solved, such as collection of tissue parts, selection of buffer cleaning liquid, enzyme digestion and determination of differential centrifugation schemes, selection of flow sorting stem cell protein markers, primary cell expansion culture and determination of freezing schemes, and determination of induction medium differentiation schemes, are urgently solved. And because of anatomical specificity and heterogeneity of adipose tissue around the vessels of thoracic and abdominal aorta, the existing preparation process is likely to generate various problems such as inaccurate adipose tissue acquisition part, long time for separation and purification steps, excessive or insufficient adipose tissue digestion, easy pollution of primary cell in-vitro culture, low nucleated cell yield, low cell survival rate, low cell purity and yield, low adipogenic differentiation rate, difficult control, non-ideal stem cell flow type result and the like. More importantly, the existing adipogenic differentiation technology, namely the standard cocktail method for inducing adipogenic differentiation medium and differentiation scheme, is difficult to induce adipogenic differentiation of primary stem cells from fat sources, and the application of the adipogenic scheme is highly likely to cause the primary cells to lose the original characteristics and directional differentiation capability. In order to overcome the defects, the inventor establishes a primary mouse aortic perivascular adipose-derived mesenchymal stem cell separation and differentiation method which has high yield, high purity, controllable differentiation, stable property and better fitting with the phenotype and the property of the tissue part, has important significance in researching and screening small molecular targets of drugs for potential treatment of cardiovascular diseases, and also provides corresponding technical support for clinical research of vascular pathology.

In the existing manufacturing process, the technical means for in-vitro separation, culture and differentiation of the primary precursor mesenchymal cells derived from the adipose tissues are very lacking, the process is complex, time-consuming and labor-consuming, the production is multiplied, and the primary cells obtained after the actual operation are not ideal in terms of yield, purity, activity, differentiation rate and the like. Due to the special dig-off study and heterogeneity of the adipose tissue around the blood vessels of the thoracic and abdominal aorta of the mice (tPVAT is the adipose tissue around the blood vessels of the thoracic aorta; aPVAT is the adipose tissue around the blood vessels of the abdominal aorta), the preparation process can have the problems of inaccurate collection position, insufficient enzymolysis and digestion fat, easy pollution in vitro culture, inaccurate selection of sorting protein markers, low cell yield and purity, low adipogenic differentiation rate and the like.

The prior adipogenic differentiation technology aiming at precursor mesenchymal stem cells, namely a standard cocktail method induced adipogenic differentiation scheme is generally used for 3T3-L1 white adipose precursor cell strains which are easy to differentiate, but the adipogenic differentiation of primary mouse adipose mesenchymal precursor cells is difficult to induce efficiently and effectively. In order to better fit the brown beige fat characteristics and phenotype of the adipose tissue around the aortic blood vessel in situ and lead the adipose tissue to be directionally induced into fat, a new fat-forming stimulation drug and drug proportion are required to be tried on the basis of the original differentiation scheme. In order to solve the problems existing in the prior art, the invention aims at the problems possibly occurring in the practical process, and attempts are made to form and disclose a primary mouse aortic perivascular adipose-derived mesenchymal precursor stem cell separation and differentiation method which is relatively perfect, short in time consumption, strong in operability, efficient in differentiation and more suitable for the phenotype and the property of a tissue part for the first time through multiple practical efforts, so that the invention is completed.

Disclosure of Invention

In view of the drawbacks of the prior art, an object of the present invention is to provide a method for separating and differentiating mesenchymal precursor cells of animal fat origin.

A method for isolating and differentiating mesenchymal precursor cells of animal fat origin, comprising:

1) Firstly, primarily separating animal fat to obtain a mixed cell population containing mesenchymal precursor cells, and then separating and purifying the mixed cell population by using a flow separation marker to obtain mesenchymal precursor cells from animal fat;

2) The method comprises the steps of firstly culturing mesenchymal precursor cells from animal fat, then performing contact inhibition, then adding an induced adipogenic differentiation medium for culturing, observing cell morphology under a microscope, and when the cell morphology is observed to be normal and tiny lipid droplets appear and are positioned in cytoplasm of the cells, adding a maintenance differentiation medium for culturing, and performing identification and detection after differentiation is completed, so as to complete differentiation.

In the step 1), the flow separation marker is Pdgfra protein or/and Sm22a protein.

The amino acid sequence of the Pdgfra protein is shown as SEQ ID NO. 1. The Sm22a protein has an amino acid sequence shown in SEQ ID NO. 2.

In the step 2), the animal fat source mesenchymal precursor cells are cultured for 2-3 days and then are subjected to contact inhibition for 2-3 days. Further preferably, the animal fat-derived mesenchymal precursor cells are contact-inhibited for 2 days after 2 days of culture.

Adding the induced adipogenic differentiation medium for culturing for 3-5 days. Further preferably, the adipogenic differentiation-inducing medium is added for 4 days.

In the step 2), the step of adding the induced adipogenic differentiation medium and the step of maintaining the differentiation medium are all replaced by corresponding culture solutions every day or every two days.

In the step 2), the adipogenic differentiation induction culture medium consists of the following components: DMEM high sugar medium, FBS, dex, IBMX, insulin, indomethacin and 3,3',5' -triiodo-L-thyronine;

the dosage of the FBS is 8-12% of the mass of the DMEM high-sugar culture medium;

the dosage of Dex is 0.5-2 mu mol of the volume of the DMEM high-sugar culture medium: 1L; the method comprises the steps of carrying out a first treatment on the surface of the

The volume ratio of the IBMX to the DMEM high-sugar culture medium is 0.3-0.8 mmol:1L;

the volume of the insulin and DMEM high-sugar culture medium is 15-25 nmol:1L;

the volume of the indomethacin and DMEM high-sugar culture medium is 100-150 nmol:1L;

the volume of the 3,3',5' -triiodo-L-thyronine is 0.5-2 nmol of the volume of the DMEM high-sugar culture medium: 1L.

Most preferably:

the using amount of the FBS is 10% of the mass of the DMEM high-sugar culture medium;

the dosage of Dex is 1uM of the volume of the DMEM high-sugar culture medium;

the volume ratio of IBMX to DMEM high-sugar culture medium is 0.5mmol:1L;

The insulin and DMEM high-sugar culture medium volume of 20nmol:1L;

the volume of the indomethacin and DMEM high-sugar culture medium is 125nmol:1L;

the 3,3',5' -triiodo-L-thyronine is 1nmol of the volume of the DMEM high-sugar culture medium: 1L.

Adding the maintenance differentiation medium for culturing for 4-8 days. The differentiation maintaining culture medium consists of the following components: DMEM high sugar medium, FBS, insulin and 3,3',5' -triiodo-L-thyronine;

the insulin is 15-25 nmol of the volume of the DMEM high-sugar culture medium: 1L;

Most preferably:

the insulin is 20nmol of the volume of the DMEM high-sugar culture medium: 1L;

The aim of the invention is achieved by the following technical scheme:

the first aspect of the invention discloses a method for separating, culturing and freezing mesenchymal precursor stem cells of a rat aortic perivascular origin for the first time, the obtained nucleated cells have high yield, short separation time and high cell survival rate, and the passage can reach more than 10 generations, and the method specifically comprises the following steps:

1) Collecting adipose tissue: the mice were euthanized by intraperitoneal injection with a physiological saline solution of pentobarbital, and several mice were dissected in a sterile super clean bench and collected as adipose tissue around the vessels of the thoracic and abdominal aorta, and as adipose tissue around the white epididymis of the control group;

step 1) selecting 6-8 weeks of mice, wherein the concentration of the pentobarbital physiological saline solution is 70ug/g, and taking every 5 mice as one sample under a stereoscopic vision to collect tissues;

2) Cleaning tissue: washing the collected adipose tissues with a physiologically compatible buffer solution, and removing superfluous blood clots, broken fat, impurities and the like under a microscope;

step 2) the physiologically compatible buffer solution was KRB buffer solution (Krebs-Ringer bicarbonate buffer) with pH 7.4 by aseptic filtration with 1% -3% of penicillin (10,000 units/ml penicillin and 10,000ug/ml streptomycin) added. The KRB buffer solution comprises the following components: 129mM sodium chloride, 5mM sodium bicarbonate, 4.8mM potassium chloride, 1.2mMKH 2PO 4,1.0mM calcium chloride, 1.2mM magnesium sulfate, 10mM HEPES,0.1% BSA.

3) Enzymatic hydrolysis of adipose tissue: adding the preheated type I collagenase solution into adipose tissues according to a proper proportion, uniformly mixing and placing the adipose tissues on a constant-temperature rotor at 37 ℃, and rotating and incubating until the tissues are fully digested;

The enzymolysis digestion liquid in the step 3) comprises collagenase I and albumin. The digestive juice comprises the following components: 0.1% -0.5% of collagenase I, 63.47-126.94 mug/ml magnesium chloride, 1.5% -3% of albumin, 1% -3% of blue chain mycin (10,000 units/ml penicillin and 10,000ug/ml streptomycin), and the balance of DMEM culture solution, wherein the mass percentage concentration is the above. Wherein the following are used: the concentration of collagenase I was 0.1%, the concentration of magnesium chloride solution was 63.47. Mu.g/ml, and the concentration of albumin was 1.5%.

Step 3) the optimal enzymolysis scheme is as follows: adding 10mL of preheated type I collagenase enzyme liquid (800U/mL) into the adipose tissue of the mice at different parts of each 50mg of net weight for enzymolysis, uniformly mixing, rotating at 37 ℃ and incubating for 35 minutes at 75rpm until the adipose tissue is fully digested, and observing the tissue every 10 minutes to avoid the reduction of the vigor of overdigested cells; optimal enzymolysis scheme of white epididymal fat of mice: the optimal concentration of the type I collagenase enzyme solution is 300U/ml, the rotation incubation time is shortened to 30 minutes, and other methods are consistent with the scheme.

4) Differential centrifugation to obtain a heterogeneous cell pellet: adding a common standard culture medium such as DMEM (Dulbeccos modified Eagle medium) high sugar or D/F12 high sugar standard culture medium of 5% -15% Fetal Bovine Serum (FBS) to terminate enzymolysis, and separating by an average density centrifugation method to obtain a large amount of heterogeneous cell masses of adipose-derived mesenchymal precursor stem cells;

And 4) the enzymolysis stopping solution is a DMEM high-sugar standard medium of 10% FBS, and the enzymolysis stopping solution is subjected to centrifugation at 500g at 4 ℃ for 5min after digestion is stopped. And (3) after centrifugation, mixing the centrifuged liquid and the sediment by intense vortex, and centrifuging the cell suspension by the same method to obtain the mixed cell mass.

5) Purifying cell mass by removing blood cells: after centrifugation to discard the supernatant, the pellet heterogeneous cell mass erythrocyte lysate is washed and resuspended to destroy residual blood cells. Then centrifuging and adding fresh culture medium for re-suspending, and obtaining mesenchymal precursor stem cells with higher purity after repeated centrifuging and culture medium re-suspending;

6) Filtering cells and culturing: the centrifuged cells were filtered with a cell filter screen, and the filtrate was centrifuged. After centrifugation, the supernatant is discarded, the cells are added with a preheated fresh culture medium to be resuspended, and then the mixture is placed in a culture dish to be cultured in a saturated humidity cell incubator with 5% carbon dioxide concentration at a constant temperature of 37 ℃, and the morphology of primary mesenchymal precursor stem cells of fat sources around blood vessels of thoracic and abdominal aorta after culture passage is shown in the figure 1;

step 6) the cells are respectively filtered by a 100um cell sieve and a 40um cell sieve in sequence, the primary cell culture dish is coated with 0.1% gelatin for more than 30 minutes, and the cell culture effect is optimal after the primary cell culture dish is preheated.

7) And (3) performing expansion culture: after the 24 th hour of primary cell culture, the culture solution in the dish is completely replaced, the non-adherent cells are removed by washing twice with PBS solution, fresh culture medium is added, and after the cells are completely grown, the cells are subjected to passage expansion culture. Washing cells by PBS, adding pancreatin to digest primary precursor stem cells at 37 ℃ for a plurality of minutes, adding fresh culture medium to stop digestion, collecting cell suspension, centrifuging to remove upper liquid, adding fresh culture medium to mix evenly and re-suspend, inoculating the suspension to a plurality of dishes, and culturing in a cell culture box until the cells grow fully;

8) Flow sorting and identification: the primary cells which are expanded and cultivated are digested and collected by a pancreatin digestion method, the collected primary cells are marked by corresponding flow type antibodies according to cell surface marking proteins of adipose-derived mesenchymal precursor stem cells around blood vessels of thoracic and abdominal mice in the literature, and the cells after separation are the high-purity adipose-derived mesenchymal precursor stem cells around blood vessels of the aorta by using a flow cytometer. Culturing the flow-sorted cells for two generations in the 7) expansion culture mode, and identifying corresponding cell surface marker proteins by using an analytical flow cytometer with a small amount of expanded primary stem cells, wherein the specific surface markers of the mesenchymal stem cells of tPVAT source are selected from Pdgfra and Sm22a of aPVAT source, and the flow identification result is shown in figure 2;

9) Primary cell viability and endotoxin detection: performing pancreatin digestion and collection on the primary cells expanded in the step 8), and using the supernatant after centrifugation as detection of intracellular toxins; the collected cell sediment is resuspended by a proper amount of culture solution, and the cell quantity and the cell activity are detected;

10 Passaging and cryopreserving: and (3) re-suspending the primary cells expanded in the step (8) by using a freezing solution, uniformly mixing the cell suspension, transferring and sub-packaging the cell suspension into a commercial freezing tube, sealing a cover and attaching a note, rapidly transferring the cell suspension into a program cooling instrument, transferring the cell suspension into a warehouse to be detected after the temperature is cooled to-80 ℃, and transferring the cell suspension into the cell warehouse for long-term liquid nitrogen low-temperature storage after the detection result is qualified.

Step 10) the optimal cryopreservation protocol is 8×10 for primary adipose mesenchymal stem cells ⁶ cells/tubes were frozen with serum-free stem cell frozen stock. The fat stem cell cryopreservation liquid comprises the following components: the concentration of the serum-free frozen stock solution is 90-95%, the concentration of the serum substitute HELIOS is 5-10%, and the above are in volume percent. The serum-free frozen stock solution is CELLBANCER serum-free frozen stock solution.

(2) The third aspect of the present invention discloses for the first time a lipid-forming induced differentiation scheme of adipose-derived mesenchymal precursor stem cells around aortic blood vessels of mice, specifically comprising the following steps:

1) Plating and culturing of mesenchymal precursor stem cells of perivascular fat source of aorta of mice: the mesenchymal stem cells obtained by the method are paved in a pore plate with 6 holes or 12 holes or smaller, so that the growth and fusion of the paved cells to more than 90% in 48 hours are ensured, and the differentiation is started after the whole replacement fresh culture medium is replaced and continuously cultured in an incubator for a plurality of days.

Step (2) 1) the mesenchymal stem cells are according to about 1X 10 ⁶ cell/cell concentration per well was plated in 6-well plates or according to 5X 10 ⁵ The cell concentration of the cell/each hole is paved in a 12-hole plate (the higher the differentiation efficiency of the minimum hole plate), the growth and fusion of the paved cells in 48 hours are ensured to be inhibited by contact, the whole fresh culture medium is replaced to continue to be cultured in an incubator for 48 hours, the differentiation is optimized after 48 hours after the contact inhibition of the cells, and then the differentiation culture medium is added to induce the cells to form lipid.

2) Selection of induced adipogenic differentiation medium composition of adipose-derived mesenchymal precursor stem cells around the aortic blood vessel of mice:

the prior art induced adipogenic differentiation medium is generally prepared by mixing a DMEM high-sugar standard medium containing 5% -15% FBS with a certain concentration of corticosteroids dexamethasone (Dex), phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) and insulin, wherein the working concentrations of the three drugs are generally as follows: 1uM Dex, 0.5mM IBMX and 1ug/ml insulin. The differentiation maintenance medium is generally DMEM high glucose standard medium of 5% -15% fbs plus a concentration of insulin, typically acting at a concentration of 1ug/ml. The cocktail method culture medium can induce white fat precursor cell strains, but is difficult to induce the adipogenic differentiation of primary stem cells of fat sources, and the application of the adipogenic scheme can greatly lead the primary cells to lose the original characteristics and the directional differentiation capacity of the primary cells, so that the mesenchymal precursor cells of fat sources around the blood vessels of primary aorta of mice are subjected to efficient and effective adipogenic differentiation.

The invention discloses the composition and the differentiation scheme of the differentiation medium for the first time, so that the differentiated cells are more attached to the characteristics and the phenotype of the adipose tissue around the blood vessel of the aorta of the mouse.

In the invention, the fat around the thoracic and abdominal aorta corresponds to brown fat phenotype and beige fat phenotype, and the induced adipogenic differentiation culture medium of the two types of fat comprises the following components: 10% FBS, DMEM high sugar medium, 1uM Dex, 0.5mM IBMX, 20nM insulin, 125nM indomethacin (Indomcin) and 1nM 3,3',5' -triiodo-L-thyronine (T3); the maintenance differentiation medium was 10% FBS, DMEM high glucose medium, 20nM insulin and 1nM T3; the epididymal white fat induction adipogenic differentiation medium is 10% FBS, DMEM high sugar medium, 1uM Dex, 0.5mM IBMX, 20nM insulin and 1uM rosiglitazone; the maintenance differentiation medium was 10% FBS, DMEM high glucose medium, 20nM insulin and 1uM rosiglitazone.

3) Adipogenic differentiation protocol: after primary cells are plated and cultured for 2 days to inhibit 2 days, adding a adipogenic differentiation induction culture medium for culturing for 4 days, observing the cell morphology under a microscope, and when the cell morphology is observed to be normal and tiny lipid droplets appear and are positioned in cytoplasm of the cells, adding a differentiation maintenance culture medium for 4-6 days, fully replacing corresponding culture solutions in two days, and carrying out identification and detection after differentiation is completed. The cell morphology of the mesenchymal precursor stem cells derived from adipose tissue around the vessels of the thoracic and abdominal aorta at different periods of differentiation is shown in fig. 3.

After the mature adipocytes become more in the late differentiation stage in the step (2) and 3), the cell layers are easy to curl and float, so that the sample is lost, the old culture liquid is required to be gently sucked away when the liquid is changed, and the gun head is prevented from directly touching the cell layers; the liquid is slowly added from the side wall by a liquid transfer device when fresh culture liquid is added, so that the liquid is prevented from directly impacting the cell layer. The induced adipogenic differentiation medium is cultured for not more than 4 days after the cells are added, and the induced adipogenic differentiation medium is cultured for not more than 6 days after the cells are added, otherwise, the differentiated cells are easy to float due to poor state.

4) Identification of morphology related to adipogenic differentiation: the differentiated primary cells were stained with oil red O, and cell morphology was observed under a microscope for cell phenotype identification. The formation of intracellular oil droplets was observed 4 days after the addition of the induction differentiation medium, and the increase of intracellular oil droplets was continued with the addition of the induction differentiation medium. According to the related literature, different types of fat cells can be identified in a phenotype mode according to the size distribution of oil drops in cytoplasm of the cells, the differentiated mature white fat cells contain a plurality of single-chamber lipid drops with larger volumes, the cells are polygonal, and cell nuclei are positioned at the edges of the cells and contain a small quantity of mitochondria. While the fat droplets of white adipocytes are single-chamber and have a large droplet volume, brown or beige adipocytes contain multi-chamber droplets of lipid, distributed throughout the cytosol of the cell, and are rich in mitochondria. The mesenchymal precursor stem cells from adipose tissue around the vessels of the thoracic and abdominal aorta were differentiated and stained with oil red O, and the cell phenotype results are shown in FIG. 4.

5) Detection of adipogenic differentiation-related protein markers: and detecting the cell adipogenic differentiation related protein marker by using a real-time fluorescent quantitative nucleic acid amplification qPCR method and a Western blot method.

The step (2) and 5) of the cell adipogenic differentiation related protein marker means that when the precursor adipose stem cells are differentiated towards the direction of white adipose cells, white adipose marker genes such as PPARa, PPARy, adiponectin, fabp genes are highly expressed; in the differentiation towards brown adipocytes, in addition to the high expression of mature fat genes similar to white fat, brown fat marker genes such as Dio2, prdm16, PGC1- α, elovl3, cox7a1 and Ucp1 are also highly expressed. When the precursor cells are differentiated towards the beige adipocytes, the mature fat genes similar to white fat are highly expressed, and the beige fat marker genes such as Tbx1, slc27a1, CD40, CD137 and CITED1 are also highly expressed.

Compared with the prior art, the invention has the following advantages:

1. the specific KRBS buffer solution is used for replacing normal saline to preserve and clean adipose tissue samples, so that adipose tissue is protected without affecting the yield of precursor stem cells.

2. In 2019, published literature, "Single-Cell RNA-Sequencing and Metabolomics Analyses Reveal the Contribution of Perivascular Adipose Tissue Stem Cells to Vascular Remodeling" (Gu W, nowak WN, xie Y, le Bras A, hu Y, deng J, issa Bhaloo S, lu Y, yuan H, fidanis E, saxena A, kanno T, mason AJ, dulak J, cai J, xu Q.Single-Cell RNA-Sequencing and Metabolomics Analyses Reveal the Contribution of Perivascular Adipose Tissue Stem Cells to Vascular Remodling.Arteriosler Thromb Vasc biol.2019Oct;39 (10): 2049-2066.) the perivascular fat of mice was digested with PBS buffer and high concentration type I collagenase only, and the tissue was digested with a high-speed rotor for a long period of time, consuming the tissue samples and easily over-digested to reduce the precursor stem Cell yield and activity. The method uses KRB buffer solution to attach to the physiological buffer environment of the adipose tissue, uses 0.1% type I collagenase digestive enzyme with low concentration to digest the adipose tissue, adds 1.5% bovine serum albumin to protect cells from over digestion, and adds enzyme activator magnesium chloride to promote digestion of digestive enzyme. The method only takes 35 minutes to digest tissues, and the method adds bovine serum albumin for enzymolysis to protect cells, so that the survival rate of the obtained precursor adipose-derived stem cells is 96.2+/-3 percent, and the obtained nucleated cells are more than those obtained by the method in the literature.

3. The separated fat precursor mixed cell mass is added into erythrocyte lysate to lyse redundant residual erythrocyte, and mesenchymal fat precursor stem cells with higher purity can be rapidly obtained after repeated centrifugation and culture medium resuspension are carried out. Compared with the traditional method that the tissue is sucked by using the sedimentation principle of the tissue and the red blood cells to remove the settled red blood cells after the tissue is uniformly mixed by using the normal saline, the time is shorter, and the obtained stem cells are purer.

4. In 2018 published literature Improved GMP compliant approach to manipulate lipoaspirates, to cryopreserve stromal vascular fraction, and to expand adipose stem cells in xeno-free media, the cell viability after cryopreservation was only: 69.0+/-3.2%, and the method uses serum-free frozen stock solution to add serum substitutes, so that the survival rate of the frozen cells can reach 96+/-4%.

5. In the prior art Chinese patent 'method for clinical purification, separation, culture and expansion and freezing storage of adipose-derived mesenchymal stem cells', primary adipose precursor stem cells are directly obtained from adipose tissues, but precursor adipose stem cells which are digested and collected by pancreatin after expansion and culture are subjected to cell sorting by a flow cytometer according to surface marker proteins Sm22a and PDGFRa of adipose cells around the blood vessels of thoracic and abdominal mice in the literature, and a large number of high-purity adipose-derived mesenchymal precursor stem cells around the blood vessels of the mice are obtained after the cell sorting.

6. In the prior art Chinese patent 'a method and application of inducing differentiation of adipose-derived stem cells into adipocytes', mature adipocytes were obtained by inducing differentiation of adipose precursor stem cells for 7-15 days using DMEM medium containing 10% FBS, 0.5mM isobutyl-methylxanthine IBMX, 1uM dexamethasone, 10uM insulin, 200uM indomethacin. While this method induced differentiation of precursor stem cells into two-step cocktail differentiation, precursor stem cells were first induced to differentiate for 4 days with DMEM high sugar medium composed of 10% fbs, 1uM dexamethasone Dex, 0.5mM IBMX, 20nM insulin, 125nM indomethacin (indomethacin) and 1nM 3,3',5' -triiodo-L-thyronine (T3); and then the precursor stem cells are maintained to differentiate for 4 days by using a DMEM high sugar culture medium with the components of 10% FBS, 20nM insulin and 1nM T3, and the total differentiation and maintenance only need 8 days to obtain mature fat cells, and the differentiation rate of the precursor stem cells can reach more than 97% by using the method. The differentiation scheme of the invention aims at brown beige fat characteristics of adipose tissue around the blood vessel of the aorta of the mouse, such as characteristics of multiple mitochondria, multiple atrial lipid droplets, multiple expression thermogenesis proteins and the like, thyroid hormone triiodothyronine (T3) is added to promote electron transfer in adipocytes and synthesis uncoupling of ATP in mitochondria to activate thermogenesis by increasing thermogenesis protein UCP1 protein, thereby not only improving the adipogenic differentiation rate of adipose precursor stem cells, but also enabling the precursor stem cells to differentiate into mature brown or beige adipose cells with perivascular adipose cell surface proteins. The adipose-derived stem cells prepared by the invention can be directly applied to clinical tests.

Compared with the prior art, the tissue enzymolysis scheme of the invention is simpler, and the operation production process, purification, separation and culture amplification processes are simplified. The method can remarkably improve the cell yield, purity, cell activity and cell property of the mesenchymal precursor cells of the perivascular fat source of the aorta of the mouse, and effectively reduce the cost of cell preparation. The invention forms a high-efficiency adipogenic induction differentiation scheme aiming at the characteristics and heterogeneity of adipose tissue around the aortic blood vessel of the mouse, thereby greatly improving the adipogenic differentiation rate of the primary cells of the mouse, leading the primary cells of the mouse to differentiate into mature adipocytes which are most in line with the anatomical in-situ property of the tissue and meeting the clinical scientific research requirements.

Drawings

Other features, objects and advantages of the present invention will become more apparent upon reading of the detailed description of non-limiting embodiments, given with reference to the accompanying drawings in which:

FIG. 1 is a morphological diagram (X100 times) of mesenchymal precursor cells of fat origin around the primary aortic blood vessel of a mouse;

a. primary mesenchymal precursor stem cells derived from perivascular fat of the thoracic aorta;

b. primary mesenchymal precursor stem cells derived from perivascular fat of abdominal aorta;

FIG. 2 is a flow chart of a mesenchymal precursor stem cell from perivascular fat source of a primary aorta of a mouse;

FITC negative control; PE negative control; apc negative control;

af 647 negative control; pb450 negative control; PE-Cy7 negative control;

cd34 expression negative; cd31 expression negative; CD19 expression negative;

CD45 expression negative; cd73 expression positive; cd90 expression positive;

cd146 expression positive; pdgfra expression positive (tpat); sm22a expression positive (avat);

FIG. 3 is a morphology of adipogenic differentiation of mouse primary aortic perivascular adipose-derived mesenchymal precursor stem cells;

a. adipose-derived mesenchymal precursor stem cells around the blood vessels of the thoracic aorta are subjected to adipogenic differentiation culture for four days, and small lipid droplets begin to appear in a few cells;

b. adipose-derived mesenchymal precursor stem cells around the blood vessels of the thoracic aorta are subjected to adipogenic differentiation culture for six days, and lipid droplets appear in the cells;

c. adipose-derived mesenchymal precursor stem cells around the blood vessels of the thoracic aorta are subjected to adipogenic induction differentiation culture for eight days, and the cells are filled with multi-chamber lipid droplets;

d. adipose-derived mesenchymal precursor stem cells around abdominal aorta vessels were cultured for four days in adipogenic induction differentiation, and small lipid droplets began to appear in a few cells;

e. adipose-derived mesenchymal precursor stem cells around abdominal aorta vessels are subjected to adipogenic differentiation culture for six days, and lipid droplets appear in the cells;

f. Adipose-derived mesenchymal precursor stem cells around abdominal aorta vessels are subjected to adipogenic induction differentiation culture for eight days, and cells are filled with multi-chamber and single-chamber lipid droplets;

FIG. 4 is a graph of oil red O staining of mice primary adipose-derived mesenchymal precursor stem cells after adipogenic differentiation;

a. a differentiation staining chart of mesenchymal precursor cells of fat source around the blood vessel of the thoracic aorta;

b. a map of differential staining of mesenchymal precursor stem cells of perivascular fat origin of the abdominal aorta;

FIG. 5 is a graph showing transcriptional level detection of genes involved in adipogenic differentiation of primary adipose-derived mesenchymal precursor stem cells in mice;

a. perivascular adipose-derived mesenchymal precursor cells of thoracic aorta (black columns) were subjected to post-adipogenic differentiation (gray columns) gene transcript levels;

b. lipid-forming specific differentiation (gray columns) gene transcription level of perivascular adipose precursor cells of thoracic aorta (contrast: adipose-derived mesenchymal precursor cells around epididymis after lipid-forming differentiation (black columns));

c. perivascular adipose-derived mesenchymal precursor cells of the abdominal aorta (black columns) were subjected to post-adipogenic differentiation (gray columns) gene transcription;

d. lipid-forming specific differentiation (gray columns) gene transcription level of adipose precursor cells around abdominal aorta (lipid-forming differentiation followed by epididymal adipose-derived mesenchymal precursor cells (black columns);

FIG. 6 is a test of the expression of a mouse primary adipose-derived mesenchymal precursor stem cell adipogenic differentiation-related protein;

a. expression level of adipose-derived mesenchymal precursor stem cells around the blood vessels of the thoracic aorta (tvataads) to adipogenic differentiation-related key protein (control is adipose-derived mesenchymal precursor cells around epididymis (eWATADSCs) to adipogenic differentiation-related key protein);

b. the expression level of adipose-derived mesenchymal precursor stem cells (aPVATADSCs) around abdominal aorta (control is adipose-derived mesenchymal precursor cells around epididymis) is the adipose-derived mesenchymal precursor stem cells.

Detailed Description

The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the present invention, but are not intended to limit the invention in any way. It should be noted that variations and modifications could be made by those skilled in the art without departing from the inventive concept. These are all within the scope of the present invention. The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. Experimental methods for which specific conditions are not noted in the examples below, all reagents and animals in the case of the present invention are commercially available according to conventional methods and conditions, or selected according to the commercial specifications. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.

Before the method for separating and differentiating the adipose-derived precursor mesenchymal stem cells around the aortic blood vessels of the mice is implemented, the mice are required to be cultured and prepared in a laboratory, and the specific working contents are as follows:

1. purchase and feeding of laboratory animals

SPF grade C57BL/6 mice, 18-22g, males, animals were supplied by Shanghai, southern model animal center. The mice are fed in separate cages, 4-5 mice per cage are fed in laboratory animal centers of Zhejiang university, the air conditioner controls the room temperature to be 23-26 ℃ and the relative humidity to be within 55+/-10%, the lighting is implemented in a 12-hour day-night period, and the ingestion and drinking water are freely carried out. The animal management and use flow accords with the national standard of experimental animal use and management, and is approved by the Fuli ethics examination committee of the Zhejiang university experimental animal.

2. Experimental medicine and reagent

(1) Surgical instrument: surgical instruments such as surgical pointed forceps, surgical dissecting forceps, surgical straight elbow scissors, dissecting elbow forceps, fine surgical forceps, spring scissors and the like are prepared and autoclaved before the dissecting operation.

(2) Preparing KRB buffer washing solution: 118mM NaCl, 5mM KCl, 2.5mM CaCl2, 2mM KH2PO4, 2mM MgSO4, 25mM NaHCO3 and 5mM glucose are dissolved in double distilled water, and simultaneously a proper amount of penicillin-streptomycin diabody is added, the pH value is adjusted to 7.4, water is added to fix the volume to 1L, and the mixture is filtered and sterilized to 0.22um and stored at the temperature of minus 20 ℃.

(3) Preparing a digestive thoracic and abdominal aorta perivascular adipose tissue enzyme solution: dissolving 0.1% type I collagenase in KRB buffer solution, adding 2mM Cacl2 and 1.5% Bovine Serum Albumin (BSA), sterilizing by filtration with 0.22um filter after the solid is completely dissolved, and preheating in 37 ℃ water bath for 1 hour; for digestion of white epididymal fat (as a control), the type I collagenase concentration was 0.05%.

(4) Digestion stop solution: DMEM high sugar medium of 10% fetal bovine serum FBS with 1X double antibody solution was added.

(5) Erythrocyte lysate: solutions were purchased from Biyun biotechnology Co.

(6) Gelatin coating solution: 1g of gelatin solid was dissolved in PBS buffer to prepare a 0.1% gelatin solution.

(7) Flow-through antibody: FITC, PE, APC, AF 647, PE-Cy7, PB450, CD34, CD31, CD19, CD45, CD73, CD90, CD146, PDGFRa and SM22a antibodies were purchased from eBioscience and abcam corporation;

(8) Frozen stock solution: serum-free stem cell frozen stock solution is preserved at a low temperature of 4 ℃.

(9) 2.5mM dexamethasone stock (2500X) preparation: 10mg of the extract was dissolved in 10ml of absolute ethanol, and the extract was filtered and sterilized and stored at-20 ℃.

(10) 1mM IBMX stock solution (500X) preparation: 55.6mg was dissolved in 1ml of 0.35M potassium hydroxide solution, and the solution was filtered and sterilized and stored at-20 ℃.

(11) 125mM indomethacin stock (1000X) preparation: 10mg was dissolved in 1ml of DMSO, diluted 10-fold with 2.5% sodium bicarbonate solution, sterilized by filtration and stored at-20 ℃.

(12) 1mg/ml insulin stock solution (1000X) preparation: 1mg/ml of the solution was dissolved in 0.01M diluted hydrochloric acid solution, and the solution was filtered and sterilized and stored at-20 ℃.

(13) 1uMT stock solution (1000X) preparation: 1mg/ml was dissolved in 1M sodium hydroxide solution as a mother liquor, and 10ml of fresh complete medium was added to each 6.5ul of mother liquor for dilution, and the resulting solution was stored at-20℃by filtration sterilization.

(14) 1mM rosiglitazone stock (1000X) configuration: 10mg/ml was dissolved in DMSO as a stock solution, and 1.93ml fresh complete medium was added to each 71.4ul of stock solution for dilution, and 0.22um was filtered for sterilization and stored at-20 ℃.

(15) Primary cell adipogenic differentiation medium of adipose tissue origin around brown beige aortic blood vessel: 10% FBS, DMEM high glucose medium, 1 Xdiabody, 1uMDex, 0.5mM IBMX, 20nM insulin, 125nM indomethacin (Indomin) and 1nM 3,3',5' -triiodo-L-thyronine (T3); it maintained differentiation medium at 10% FBS, DMEM high glucose medium, 20nM insulin and 1nM T3.

(16) Primary cell adipogenic differentiation medium derived from epididymal white adipose tissue: 10% FBS, DMEM high sugar medium, 1uMDex, 0.5mM IBMX, 20nM insulin and 1uM rosiglitazone; the maintenance differentiation medium was 10% FBS, DMEM high glucose medium, 20nM insulin and 1uM rosiglitazone.

(17) Preparing an oil red O dye liquor: 0.2g of oil red O was added to 100ml of isopropyl alcohol, dissolved at 56℃for 1 hour, cooled and filtered as a stock solution. When in use, the storage solution and double distilled water are evenly mixed according to the proportion of 6:4, and the working solution is obtained after standing for 10 min.

(18) Preparing a cell fixing solution: the 4% pfa paraformaldehyde solution was purchased from bi yun biotechnology limited.

(19) qPCR primers: synthesized by Hangzhou Shangya Biotechnology Co., ltd, was prepared with DEPC water at a concentration of 10mM and stored at-20℃with the following primer sequences:

fabp4 gene F ':5'-GTCCT GGTACATGTGCAGAA-3';

R’:5′-CTCTTGTAGAAGTCACGCCT-3′；

the Adipoq gene is F ':5'-ATGACGACTGCCATCCTAGAG-3';

R’:5′-GCTCCCTAAAGAGCTGGGG-3′；

ucp1 gene F ':5'-GTGAACCCGACAACTTCCGAA-3';

R’:5′-TGCCAGGCAAGCTGAAACTC-3′

the beta-actin gene is F ':5'-CAGATGCCACTACAGCACG-3';

R’:5′-CCTGCCGCTGCCATAGAAG-3′

ppara gene F ':5'-CCCAAGGGAGGAATAGCTTCT-3';

R’:5′-CTCTGCGATGCGGTTCCAA-3′

pparg gene F ':5'-CTTGGCTGCGCTTACGAAGA-3';

R’:5′-GAAAGCTCGTCCACGTCAGAC-3′

dio2 gene F ':5'-ATGGGACTCCTCAGCGTAGAC-3';

R’:5′-ACTCTCCGCGAGTGGACTT-3′

prdm16 gene F ':5'-CCACCAGCGAGGACTTCAC-3';

R’:5′-GGAGGACTCTCGTAGCTCGAA-3′

PGC-1a gene, F ':5'-TATGGAGTGACATAGAGTGTGCT-3';

R’:5′-GTCGCTACACCACTTCAATCC-3′

the Cox7a1 gene is F ':5'-GCTCTGGTCCGGTCTTTTAGC-3';

R’:5′-GTACTGGGAGGTCATTGTCGG-3′

the gene Elovl3 is F ':5'-TTCTCACGCGGGTTAAAAATGG-3';

R’:5′-GGCCAACAACGATGAGCAAC-3′

the Tbx1 gene is F ':5'-TCTCGCCTGCTTAACGTGG-3';

R’:5′-CCAGCCCTGTGACACTAATCTT-3′

The Slc27a1 gene is F ':5'-CTGGGACTTCCGTGGACCT-3';

R’:5′-TCTTGCAGACGATACGCAGAA-3′

CD40 gene F ':5'-TGTCATCTGTGAAAAGGTGGTC-3';

R’:5′-ACTGGAGCAGCGGTGTTATG-3′

CITED1 gene, F ':5'-ACTAGCTCCTCTGGATCGACA-3';

R’:5′-GACCCAGTTTTGCATGGGC-3′

(20) Western blot experiment: (1) preparation of cell lysate: a 0.6%Triton X100 Tris-Hcl solution at pH 7.4 was prepared and a dose of proteasome inhibitor was added. (2) Antibody purchase: protein antibodies such as beta-action, CITED1, UCP1, pparg, ppargc1a, fabp4, adipoq and the like were purchased from abclon, CST.

Hereinafter, the present invention will be described in more detail and in detail with reference to examples, but the following examples are not intended to limit the present invention.

EXAMPLE 1 isolation and culture of adipose-derived mesenchymal precursor Stem cells around aortic blood vessels of mice

1) Collecting a adipose tissue sample: euthanasia was performed by intraperitoneal injection of 6-8 week old male C57/BL mice with a physiological saline solution of pentobarbital at a concentration of 70 ug/g. Mice were euthanized, sterilized by soaking in 75% alcohol for two minutes and dissected in a sterile superclean bench. The dissecting and collecting instruments are all disinfected, the dissected and opened mouse abdominal cavity is observed under a body view mirror, and the fat tissues around the blood vessels of the thoracic and abdominal aorta and the fat tissues around the white gonad epididymis used as a control are collected into marked EP tubes respectively and placed on ice. Five mice were used as one tissue sample, approximately 50mg of each class of tissue sample.

2) Tissue treatment: sterile filtration is carried out, a double antibody buffer solution with the pH value of 7.4 is added, tissue cleaning is carried out on ice by using the buffer solution, after redundant impurities are removed, the tissue is sucked by sterile absorbent paper, and the tissue is mechanically chopped into 1-2mm tissue pieces.

3) Enzymatic hydrolysis of adipose tissue: adding 10mL of preheated 0.1% type I collagenase enzyme solution into every 50mg of fat tissue around the thoracic and abdominal aorta of the mice with net weight for enzymolysis, uniformly mixing, placing on a constant-temperature rotor at 37 ℃, rotating at 37 ℃ for incubation at 75rpm for 35 minutes after uniform mixing until the tissue is fully digested, and observing the tissue every 10 minutes to avoid the reduction of the activity of overdigested cells; control group: 10mL of preheated 0.05% type I collagenase enzyme solution is added into each 50mg of white epididymal adipose tissue of a mouse with net weight for enzymolysis, the mixture is placed on a constant temperature rotor at 37 ℃ for rotation at 75rpm for incubation for 30 minutes at 37 ℃ after the mixture is uniform.

4) Differential centrifugation and purification of cells: after the enzymolysis digestion is finished, adding an equal volume of 10% FBS DMEM high-sugar standard medium to stop digestion, and centrifuging the mixed solution at the rotation speed of 500g at the temperature of 4 ℃ for 5min. And (3) after centrifugation, severely vortex and mix the centrifuged liquid and sediment, then centrifuge the cell suspension by the same method, and centrifuge the supernatant to obtain a large amount of adipose-derived mesenchymal precursor stem cell impurity cell clusters. Adding the mixed cell mass into the erythrocyte lysate to destroy residual blood cells, after lysing for a plurality of minutes, centrifuging, discarding the supernatant, adding fresh culture medium for re-suspension, and obtaining mesenchymal precursor stem cells with higher purity after repeated centrifugation and culture medium re-suspension adding operation;

5) Filtering cells and culturing: after resuspension of the centrifugally purified cell pellet, the pellet was sequentially filtered through 100um and 40um cell sieves, respectively, and the filtrate was centrifuged. After centrifugation, the supernatant was discarded, the pre-heated fresh medium was added to resuspend cells and inoculated into a 0.1% gelatin coated and pre-heated petri dish for 30 minutes or longer, and the petri dish was incubated in a 37℃constant temperature 5% carbon dioxide concentration saturated humidity cell incubator. The cell morphology after culture passage is shown in figure 1.

Example 2 sorting and identification of cell surface markers of adipose-derived mesenchymal precursor stem cells around aortic blood vessels of mice

1) Flow cytometry cell sorting: expanding and culturing mesenchymal precursor stem cells of fat sources around blood vessels of thoracic and abdominal aorta of the mice, washing the cells twice with PBS for 0.05% pancreatin digestion for a plurality of minutes after the cells grow fully, centrifuging at 1000rpm at 4 ℃ for 5min, discarding the supernatant, adding 1ml PBS, and re-suspending and sub-packaging in a 1.5ml centrifuge tube. After centrifugation at 1000rpm at 4℃for 5min, the supernatant was discarded and 100. Mu.l of flow Buffer (PBS+2% FBS) was added.

Surface markers of adipose-derived mesenchymal precursor stem cells around the aortic blood vessels of mice were selected according to literature. Mu.l of antibody was added to each tube of cells and mixed well: CD34-FITC (hematopoietic Stem cell markers), CD45-FITC (hematopoietic Stem cell markers), CD19-FITC (lymphoB cell markers), CD31-FITC (endothelial cell markers), CD90-PE (mesenchymal Stem cell markers), CD73-APC (adipose-derived mesenchymal Stem cell markers), CD146-Alexa Fluor 647 (adipose-derived mesenchymal Stem cell markers), PDGFRa-Pacific blue 450 (tPVAT adipose-derived mesenchymal Stem cell markers), and SM22a-PE-Cy7 (aPVAT adipose-derived mesenchymal Stem cell markers); a blank and isotype control treated group (isotype control antibodies mouse IgG FITC, PE, APC, AF647, PB450 and PE-Cy 7) was set. After the antibody was added to the cells and mixed well, the cells were incubated at 4℃for 30min in the absence of light, the cell suspension was centrifuged at 1000rpm for 5min, the supernatant was discarded, 1ml of PBS was added for washing once, and the cells were immediately sorted by flow cytometry.

2) Identification of cell surface marker proteins by flow cytometry: the cells were collected and added with the corresponding surface marker antibody, washed with PBS, mixed with 500. Mu.l of 1% paraformaldehyde in PBS, placed at 4℃in the absence of light, and the fluorescence intensity of the cells was measured by flow cytometry over 24 hours. In the flow analysis experiment, the cell number collected by each sample is more than or equal to 10 ⁴ And (2) carrying out fluorescence parameter analysis of the related surface marks by using Flow JO software, wherein the identification result of the Flow analyzer is shown in figure 2.

The identification result of the flow analyzer shows that after the mesenchymal precursor stem cells are separated from the flow separation aiming at the adipose tissue around the blood vessel of the aorta of the mouse, the mesenchymal stem cell population with higher purity and derived from fat is obtained, and the immune cell population such as hematopoietic stem cells, macrophages, endothelial cells, granulocytes, lymphocytes and the like is reduced to the level which cannot be detected by the flow.

EXAMPLE 3 expansion and cryopreservation of adipose-derived mesenchymal precursor stem cells around aortic blood vessels of mice

1) And (3) performing expansion culture: the culture medium was completely replaced in dishes one day after the attachment of the P0 primary cell culture. The non-adherent cells and impurities were removed by washing twice with PBS solution and fresh medium was added. After the cells are all grown, carrying out cell subculture. After washing 10cm dish of primary cells with PBS, 2ml of 0.05% pancreatin was added to digest the primary cells at 37℃for 6 minutes. When observing under the mirror, the cells become round and the cells become suspended when the plane shakes the plate Digestion was terminated by adding an equal amount of fresh medium. Blowing down cells on a culture dish by a micropipette in the same direction, transferring into a 15ml centrifuge tube, centrifuging for 5min at 500g, centrifuging to remove upper liquid, adding fresh culture medium again, mixing, and suspending again to count cells, and adding the culture medium in a ratio of 5×10 ⁶ Inoculating the cell concentration of the cell/10cm culture dish to a plurality of dishes, placing the dishes in a cell culture box for continuous culture for 48 hours or more until the cells grow to be full;

2) Primary cell viability and endotoxin detection: performing pancreatin digestion and collection after primary adipose-derived mesenchymal stem cells are expanded and cultivated, and detecting intracellular toxins by taking a supernatant after centrifugation; the collected cell sediment is resuspended by a proper amount of culture solution, and the cell quantity and the cell activity are detected;

3) And (5) passage freezing: the identification of the flow detection cell surface markers is correct, and the primary adipose mesenchymal stem cells with the viability and endotoxin qualified by detection are identified by 7×10 ⁶ And (3) freezing and re-suspending the cell/tube by using serum-free stem cell freezing solution, uniformly mixing the cell suspension, transferring and sub-packaging the cell suspension into a commercial freezing tube, sealing and attaching notes (marking freezing date, batch, cell number and other information), rapidly transferring the freezing tube into a program cooling instrument, and transferring the cell into a cell bank for long-term liquid nitrogen low-temperature storage after the temperature of the freezing tube is cooled to-80 ℃ after 12 hours. Example 4 differentiation and differentiation identification of adipose-derived mesenchymal precursor stem cells around aortic blood vessels of mice induced to become lipid

(1) Induction of adipogenic differentiation protocol:

1) Cell plating: mesenchymal precursor stem cells of perivascular fat origin and periepididymal fat origin of the aorta of mice were used in 1×10 ⁶ The cell concentration of the cell/each hole is paved on a 6-hole plate, the growth and fusion of the paved cells in 48 hours are ensured to be inhibited by contact, the whole fresh culture medium is replaced and continuously cultured in an incubator for 48 hours, the differentiation is started to be optimal after 48 hours after the contact inhibition of the cells, and then the differentiation inducing culture medium is added to induce the cells to form lipid differentiation.

2) Use scheme of induced adipogenic differentiation and maintenance differentiation medium: in order to be more attached to the characteristics and phenotype of the adipose tissue around the blood vessels of the aorta of the mice, the invention tries to prepare a new proportion and adipogenic differentiation stimulus on the basis of a cocktail method differentiation scheme, prepares a culture medium for inducing adipogenic differentiation and maintaining differentiation of fat brown and beige fat around the aorta of the chest and abdomen, and induces adipogenic differentiation and maintaining differentiation of epididymal white fat. After primary adipose mesenchymal stem cells are plated and cultured for 2 days to inhibit 2 days, adding an induced adipogenic differentiation medium for culturing for 4 days, observing cell morphology under a microscope, adding a maintenance differentiation medium for corresponding type of adipose for 4-6 days when the cell morphology is normal and tiny lipid drops appear and are positioned in cell cytoplasm, and carrying out identification and detection after the differentiation is completed by fully replacing corresponding culture solutions for two days in the steps. The induced adipogenic differentiation medium is added to the cell culture for not more than 4 days, the differentiated medium is maintained to be added to the cell culture for not more than 6 days, otherwise, the differentiated cell state is worsened and is easy to float. After mature adipocytes become more in the late differentiation stage, cell layers are easy to curl and float, so that the loss of samples is caused, the liquid is required to be gentle when the liquid is changed, the old culture liquid is required to be light and slow when the old culture liquid is sucked away, and the gun head is prevented from directly touching the cell layers; the liquid is slowly added from the side wall by a liquid transfer device when fresh culture liquid is added, so that the liquid is prevented from directly impacting the cell layer. The cell morphology of the differentiation results at different stages is shown in FIG. 3.

(2) Lipid-forming differentiation-related assays

1) Morphological identification of oil red O stained cells: washing primary cells from different kinds of lipid tissues after differentiation for two times by PBS, fixing the cells in situ for 15min by 4% PFA, staining the cells for 30min at 37 ℃ by using oil red O staining working solution, washing the cells for 1s by using 60% isopropanol solution, removing redundant colors, washing the cells for several times by double distilled water, sealing the cells by liquid, and observing the cell morphology under a microscope for cell phenotype identification. The formation of intracellular oil droplets was observed 4 days after the addition of the induction differentiation medium, and the increase of intracellular oil droplets was continued with the addition of the induction differentiation medium. Phenotype identification can be carried out on different types of fat cells according to the size distribution of oil drops in cytoplasm of the cells, the differentiated mature white fat cells contain a plurality of single-room fat drops with larger volumes, the cells are polygonal, and cell nuclei are positioned at the edges of the cells and contain a small quantity of mitochondria. While the fat droplets of white adipocytes are single-chamber and have a large droplet volume, brown or beige adipocytes contain multi-chamber droplets of lipid, distributed throughout the cytosol of the cell, and are rich in mitochondria. The results of the oil red O stained cell phenotype after differentiation of the primary mesenchymal precursor stem cells of different types of fat sources are shown in fig. 4.

2) qPCR method for detecting transcriptional level of adipogenic differentiation related protein marker: the mesenchymal precursor stem cells of the primary fat source of the mice before and after differentiation are washed twice by PBS, and the total RNA of the primary cells is extracted by a salt ion adsorption column method. Reverse transcribing RNA into cDNA by using a universal reverse transcription kit, designing a specific primer of a lipid-related gene, and detecting the transcribed mRNA level of the lipid-related gene of the differentiated primary cell by using a real-time quantitative fluorescent PCR kit, wherein beta-actin is used as an internal reference. The results are shown in FIG. 5.

3) Western blot Western blotting method for detecting expression level of adipogenic differentiation related protein markers: washing the mesenchymal precursor stem cells of the primary fat source of the mice before and after differentiation twice by PBS, digesting and collecting by pancreatin, centrifuging and discarding the supernatant, collecting cell sediment, lysing cells by using cell lysate and simultaneously crushing the cells by using an ultrasonic instrument, centrifuging and collecting the supernatant to obtain the total cell protein lysate. Adding a proper amount of SDS-PAGE loading buffer solution into the protein lysate, heating the protein in a metal bath at 100 ℃ for 5 minutes, adding the obtained WB protein sample onto an SDS-PAGE gel, performing Western blot Western blotting experiment, and detecting the protein expression level of the differentiated primary cell adipogenic related gene. The WB experiment mainly detects the key proteins of PPARy, adiponectin, PGC 1-alpha, ucp1, tbx1, slc27a1 and the like, and compares the results of the formation of the key proteins corresponding to the differentiation of cells between white fat sources, and the results are shown in figure 6.

The results show that when adipose-derived mesenchymal precursor stem cells differentiate towards white fat, the cells highly express adipogenic differentiation marker genes such as PPARa, PPARy, adiponectin, fabp4 and the like, no matter at the transcription level or the protein expression level; in addition to the mature fat genes, brown fat marker genes such as Dio2, prdm16, PGC1- α, elovl3, cox7a1 and Ucp1, or beige fat marker genes such as Tbx1, slc27a1, CD40, CD137 and CITED1 are also highly expressed when differentiating in the brown or beige fat direction.

The invention discloses a scheme for separating and differentiating mesenchymal precursor stem cells of a fat source around a blood vessel of a mouse aorta for the first time, wherein the scheme simplifies a tissue enzymolysis scheme, operates a production process, improves the yield, purity and cell activity of primary cells, retains heterogeneity and anatomical specificity of the primary cells, meets the requirements of clinical research and industrial production, and effectively reduces the cost of a preparation process of the primary mesenchymal precursor stem cells of the mouse. In addition, the invention discloses a differentiation scheme for high induced adipogenic differentiation rate of mesenchymal precursor stem cells around the blood vessels of the aorta of the mice for the first time, and the differentiated cells retain anatomical characteristics and heterogeneity of adipose tissues around the blood vessels of the aorta of the mice, thereby providing corresponding technical support for research and screening of small molecular targets of drugs for potential treatment of cardiovascular diseases and clinical research of vascular pathology.

The above examples merely represent a few embodiments of the present invention, which are described in more detail and detail. The description of the embodiments is only for aiding in understanding the method of the present invention and its core ideas. The present invention is illustrated by way of example and not by way of limitation, and modifications and variations may be readily made by those skilled in the art without departing from the principles of the invention, but such modifications and variations are within the scope of the invention as defined in the appended claims.

Claims

1. A method for isolating and differentiating mesenchymal precursor cells of animal fat origin, comprising:

2. The method for separating and differentiating animal fat derived mesenchymal precursor cells according to claim 1, wherein in step 1), the flow separation marker is Pdgfra protein or/and Sm22a protein.

3. The method for isolating and differentiating animal fat derived mesenchymal precursor cells according to claim 2, wherein in step 1), the amino acid sequence of the Pdgfra protein is shown in SEQ ID No. 1.

4. The method for isolating and differentiating animal fat-derived mesenchymal precursor cells according to claim 2, wherein in step 1), the amino acid sequence of Sm22a protein is shown in SEQ ID No. 2.

5. The method for separating and differentiating animal fat-derived mesenchymal precursor cells according to claim 1, wherein in step 2), the animal fat-derived mesenchymal precursor cells are cultured for 2 to 3 days and then contact-inhibited for 2 to 3 days in step 2).

6. The method for isolating and differentiating animal fat-derived mesenchymal precursor cells according to claim 1, wherein the step 2) is performed by adding a lipid-forming differentiation-inducing medium for 3 to 5 days.

7. The method for isolating and differentiating animal fat-derived mesenchymal precursor cells according to claim 1, wherein in step 2), the step of adding the induced adipogenic differentiation medium and the step of maintaining the differentiation medium are performed by replacing the respective culture medium in total every day or every two days.

8. The method for isolating and differentiating animal fat derived mesenchymal precursor cells according to claim 1, wherein in step 2), the adipogenic differentiation-inducing medium is composed of: DMEM high sugar medium, FBS, dex, IBMX, insulin, indomethacin and 3,3',5' -triiodo-L-thyronine;

the volume of the insulin and DMEM high-sugar culture medium is 15-25 nmol:1L;

9. The method for isolating and differentiating animal fat-derived mesenchymal precursor cells according to claim 1, wherein in step 2), a maintenance differentiation medium is added for 4-8 days.

10. The method for isolating and differentiating animal fat derived mesenchymal precursor cells according to claim 1, wherein in step 2), the differentiation maintaining medium is composed of: DMEM high sugar medium, FBS, insulin and 3,3',5' -triiodo-L-thyronine;