CN115029305A

CN115029305A - Separation and identification method for pig FAPs (FAPs) cells and application of FAPs cells

Info

Publication number: CN115029305A
Application number: CN202210689455.3A
Authority: CN
Inventors: 单体中; 刘事奇; 有文静; 涂羽昂; 汪以真
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2022-06-17
Filing date: 2022-06-17
Publication date: 2022-09-09

Abstract

The invention discloses a separation and identification method of FAPs cells of a pig and application of the FAPs cells. The method comprises the following steps: 1) 1) separating and digesting the longissimus dorsi of the pig; 2) sorting the pig FAPs cells; 3) and identifying the porcine FAPs cells to obtain a specific PDGFR alpha antibody for identifying the porcine FAPs cells by immunofluorescence. The application of the obtained porcine FAPs cells, 1) the porcine FAPs obtained by separation are used for researching the formation regulation and control of porcine intramuscular fat and the molecular mechanism thereof; or 2) the gene expression rule and the gene function related to the lipid metabolism in the process of cell adipogenesis of the porcine FAPs are researched; or 3) as seed cells for cell culture meat production for intramuscular fat production in cell culture meat. The invention has important practical significance and application prospect for FAPs adipogenic differentiation fate, pork intramuscular fat deposition regulation and control, and culture and production of intramuscular fat in cell culture meat.

Description

Separation and identification method for pig FAPs (FAPs) cells and application of FAPs cells

Technical Field

The invention relates to a method for separating and detecting porcine fibroblast/adipogenic progenitor cells (FAPs) and application of the FAPs, belonging to the application technology in the fields of modern agriculture and food research.

Background

FAPs cells are a subset of mesenchymal cells located between muscle bundles and muscle fibers, and the typical molecular marker gene is platelet derived growth factor receptor alpha (PDGFR α). FAPs cells play a plurality of important physiological functions and participate in regulating the microenvironment of muscle tissues, the development of skeletal muscles, the repair of muscle injury and the like. It was found that FAPs cells are the main source of intramuscular adipocytes in the process of muscle regeneration in humans and mice, and have the potential to differentiate into adipocytes and fibroblasts. In pork production, intramuscular fat directly affects the pork quality, and how to regulate intramuscular fat deposition has important significance for high-quality pork production. Proliferation, adipogenic differentiation capacity and quantity of FAPs cells can directly influence the content of intramuscular fat of pork, but an efficient and rapid pig FAPs cell separation method is lacked at present, and a regulation mechanism of adipogenic differentiation fate of the pig FAPs cells is not reported.

As for the current research report related to the in vitro culture of the porcine intramuscular fat cells, a series of technical problems exist, such as: 1) intramuscular fat precursor cells obtained by a common differential adherence method usually contain various types of cells, and the adipogenic differentiation capacity of the cells is general, so that the application of the cells in the research fields of an intramuscular fat formation mechanism, cell culture meat and the like is limited to a great extent; 2) Due to the lack of a separation and identification method of FAPs cells in pig muscle, in vitro culture methods, effective adipogenic differentiation culture media and methods are unknown, and the differentiation polyester capacity and the availability of FAPs for gene function related research need to be further researched.

Disclosure of Invention

In order to overcome the problems in the prior art, the invention aims to provide a method for separating and identifying porcine FAPs cells and application of the FAPs cells.

FAPs cells, a separation and identification method and application thereof comprise the following steps:

1) isolation and digestion of the longissimus dorsi of pigs: taking 3-5 days old piglets, killing after anesthesia and disinfecting with alcohol, taking the longissimus dorsi in an aseptic super-clean workbench, fully shearing tissues with surgical scissors, and then according to the tissue volume: digestive enzyme volume = 1: 3 adding 0.2% collagenase I, digesting for 1 h at 37 ℃, filtering by a filter screen with 70 μm and a filter screen with 40 μm, and centrifuging for 6 min at 500 g;

2) sorting porcine FAPs cells using magnetic beads: resuspending the cell pellet obtained in 1) with a washing solution, and measuring the volume of the cell suspension: 1 × erythrocyte lysate = 1: 9, adding erythrocyte lysate, placing at 4 ℃ for lysis for 5 min, and then centrifuging for 5 min at 400 g; resuspend the cells with 1mL of 1 XBinding buffer, add 100. mu.L dead cell remover and incubate for 15 min at room temperature, then centrifuge for 5 min at 400 g; resuspend cells at 1 × Binding buffer, check cell concentration with cytometer at 2.5 μ g/10 ⁶ CD140a-Biotin antibody (130-101-905, Miltenyi Biotec) was added to each cell, incubated at 4 ℃ for 5 min, and centrifuged at 400 g for 4 min; 80 μ L of 1 × Binding buffer resuspended cells, 20 μ L of Anti-Biotin Microbeads (130-; resuspending the cells by 200 mu L of 1 × Binding buffer, passing through a magnetic column, wherein the cells staying on the adsorption column are FAPs cells of the pig, and collecting the cells;

3) immunofluorescence identification of porcine FAPs cells: inoculating the porcine FAPs cells into a 24-well plate, culturing for 3 days, performing immunofluorescence detection by adopting different PDGFR alpha antibodies after 3 days, and screening to obtain a specific antibody (A2103, ABClonal Technology) for identifying the porcine FAPs cells;

4) detecting the fat forming differentiation polyester capability and the lipid metabolism related genes of the pig FAPs: inoculating the separated FAPs into a 12-well cell culture plate for culture, inducing the FAPs to differentiate towards the adipogenic direction for 4 days by using an adipogenic induction medium (MDI medium), and then replacing the FAPs with a differentiation medium (INS medium)ium), continuously culturing for 2 days, adopting Bodipy staining to detect the condition of differentiated polyester, and detecting genes related to fat synthesis and metabolism by real-time fluorescent quantitative PCR (polymerase chain reaction) ((III)PPARg, Adipoq, FABP4Etc.); meanwhile, intramuscular precursor fat cells obtained by a differential wall pasting method are induced to differentiate, and the polyester differentiation capacities of the intramuscular precursor fat cells and the Bodipy fat cells are compared through Bodipy staining;

5) gene function study: porcine FAPs cells were transfected with GFP fluorescence-tagged SV40 lentivirus and GFP fluorescence was detected 72 hours after transfection.

The high-purity porcine FAPs cells obtained by the separation and detection method express PDGFR alpha;

the method and the application are used for researching the formation of porcine intramuscular fat cells and the production of cell culture meat, researching the regulation and control of porcine FAPs cell differentiation polyester, and also can be used for researching the expression rule and the gene function of intramuscular fat deposition related genes;

the porcine FAPs cells have good proliferation and adipogenic differentiation potential in vitro and can be used for gene function research.

The invention has the beneficial effects that:

the invention provides a method for separating and detecting the pig FAPs cells, and the pig FAPs cells obtained by means of a magnetic bead sorting technology have the advantages of convenience and rapidness compared with a flow sorting method; meanwhile, specific antibodies which can be used for pig FAPs cell magnetic bead sorting and immunofluorescence staining are determined; the pig FAPs cells are proved to have good polyester differentiation capacity through in vitro culture and adipogenic differentiation induction, and a formula for inducing the adipogenic differentiation of the pig FAPs cells is determined, so that a new method is provided for the deposition regulation of pig intramuscular fat and the research and development of cell culture meat.

The porcine FAPs cells obtained by the invention have good proliferation and adipogenic differentiation potential in vitro, are key cell models for researching the deposition of porcine intramuscular fat, can be used for researching the aspects of regulating and controlling the adipogenic and fibroblast differentiation of the porcine FAPs cells in vitro and the like, and develop a new method for researching the regulation and control of the intramuscular fat deposition and the differentiation fate of the FAPs cells at present; secondly, the separated porcine FAPs cells can be used for 3D cell culture, and the formation mechanism of intramuscular fat under in vivo conditions can be explored; in addition, the FAPs cells are the main cell source of intramuscular fat, can be used as seed cells for in vitro research and development and production of cell culture meat, and have wide application prospects.

Drawings

FIG. 1 is a technical diagram of immunomagnetic bead sorting.

Fig. 2 is a white light picture of porcine FAPs cells taken on day 1 and day 4, respectively.

FIG. 3 is a graph of results of immunofluorescent staining for detection of porcine FAPs cells.

FIG. 4 is a morphological change diagram of porcine FAPs during cell adipogenic differentiation, and is taken on

induction days

2, 4 and 6. The cells pointed by the arrows are mature intramuscular adipocytes differentiated from FAPs.

FIG. 5 shows that fat drop deposition is detected by Bodipy staining after lipogenic differentiation of pig FAPs cells obtained by magnetic bead sorting and intramuscular fat precursor cells obtained by differential adherence. The cells pointed by the arrows are mature intramuscular adipocytes differentiated from FAPs.

FIG. 6 shows the qPCR detection result of fat synthesis and metabolism key gene expression after the lipogenic differentiation of the porcine FAPs cells.

FIG. 7 is a photograph of fluorescent detection of SV40 lentivirus infected porcine FAPs cells 72 hours later.

Detailed Description

The invention will be further described with reference to the drawings and the embodiments, but the scope of the invention is not limited thereto.

(I) isolation and digestion of the longissimus dorsi of pigs

1. Preparation before isolation of Primary cells

Placing surgical scissors and scalpel into autoclave for sterilization one day in advance, and irradiating with ultraviolet lamp on superclean bench for 30 min; prepare 75% ethanol in advance, use sterile 50mL centrifuge tube to divide several tubes of 75% ethanol and PBS, and place it on ice to precool.

2. Isolation and digestion of the longissimus dorsi of pigs

1) Killing 3-5 days old piglets purchased from Hangzhou lighthouse boar Limited company by heart bloodletting, wiping the whole bodies of the piglets with 75% ethanol, transferring the piglets to a super clean bench after sterilization treatment, taking out the longissimus dorsi of the piglets, putting the longissimus dorsi of the piglets into a 50mL centrifugal tube filled with 75% ethanol, washing the piglets for several times, transferring the piglets into precooled PBS, washing the piglets twice to remove contained blood, and transferring the longissimus dorsi of the piglets into new PBS.

2) Taking out one of the small muscles, placing the small muscles in a sterile cell culture dish containing PBS, cutting the small muscles into small pieces by using a surgical scissors, transferring the small muscles into a sterile 5 mL centrifuge tube, and rapidly cutting the small muscles to 1 mm by using the surgical scissors ³ Size (c), the minced tissue was transferred to a sterile 15 mL centrifuge tube.

3) By tissue volume: digestive enzyme volume = 1: 3 Add 0.2% collagenase I (prepared from PBS solution). And (3) putting the mixed solution of the tissues and the digestive enzymes in a water bath kettle at 37 ℃ for digestion for 1 h (the specific digestion time is determined according to the actual digestion condition).

4) 1/2 volumes of serum-containing medium were added and filtered through a 70 μm, 40 μm sieve.

5) The filtrate was collected and centrifuged at 500 g for 6 min).

(II) obtaining intramuscular fat precursor cells by using a differential adherence method

And (3) resuspending the cells by using a complete culture medium, inoculating the cells to a cell culture disc, culturing for 2 hours, and then changing the culture solution to obtain the intramuscular fat precursor cells.

(III) sorting the FAPs cells by using immunomagnetic beads (as shown in figure 1)

1. Removing red blood cells

1) After centrifugation, the supernatant was removed, the cells were resuspended with 1mL of wash solution, and the cells were thoroughly blown off.

The washing liquid comprises the following components: FBS solution containing 0.1% BSA.

2) 9 mL of erythrocyte lysate (Boschd biosciences) was added, 1mL of pancreatin (Samorfei Co.) was additionally added to prevent aggregation, and the mixture was thoroughly mixed and then lysed at 4 ℃ for 4 min.

3) Centrifuge at 300g for 4 min.

2. Removal of dead cells

1) The supernatant after centrifugation was removed, the cells were resuspended in 1mL of 1 × Binding Buffer (Miltenyi Biotec), and 100. mu.L of dead cell remover (Miltenyi Biotec) was added.

2) Mixing, standing at room temperature for 15 min, and centrifuging at 300g for 4 min.

3. Incubation of PDGFR alpha (CD140a) -Biotin antibodies

1) After centrifugation, the supernatant was removed, and the cells were resuspended and blown off with 1mL of 1X Binding Buffer, and 20. mu.L of the cell suspension was taken, and the cell density was measured with a cell counter.

2) PDGFR α (CD140a) -Biotin antibody (130-.

3) Centrifuge at 300g for 4 min.

4. Incubation magnetic beads

1) The supernatant after centrifugation was removed, and the cells were resuspended in 2 mL of a washing solution and washed.

2) Centrifuge at 300g for 4 min.

3) After centrifugation, the supernatant was removed, and 1mL of 1X Binding Buffer was used to resuspend and blow off the cells.

4) mu.L of Anti-Biotin Microbeads (130. sup. 090. sup. 485. sup. Miltenyi Biotec) was added and incubated at 4 ℃ for 15 min.

5) Centrifuge at 300g for 4 min.

5. Sorting FAPs cells

1) The column was mounted (130-.

2) Subsequently, the supernatant after centrifugation was removed, and 500. mu.L of Binding Buffer was taken to resuspend and blow off the cells.

3) And sucking the cell suspension by using a 100 mu L pipette, slowly adding the cell suspension into the center of the sorting column to avoid adding the cell suspension to the side wall and generating air bubbles, and allowing PDGFR alpha negative cells to flow into a collected centrifugal tube through the sorting column, so that the cells staying in the sorting column are PDGFR alpha positive cells, namely FAPs cells.

(III) immunofluorescence identification of porcine FAPs cells

Porcine FAPs cells were seeded in 24-well plates for 3 days, followed by photographing under white light (fig. 2), observing their cell morphology, and further immunofluorescence detection using PDGFR α antibodies purchased from ABclonal Technology (a 2103), Santa Cruz Biotechnology (sc-398206), huaan organism (ET 1702-49) (fig. 3).

The results were:

as can be seen from the white light photograph, the porcine FAPs cells were spindle-shaped under planar culture conditions (fig. 2); whereas the 3 antibodies only detected PDGFR alpha protein expression in porcine FAPs cells by ABClonal Technology (A2103, ABClonal Technology), the immunofluorescence staining results showed that the cells isolated by the present invention were indeed porcine FAPs cells (FIG. 3).

(IV) research on adipogenic differentiation function and lipid metabolism related gene expression of porcine FAPs (FAPs) cells

1) The porcine FAPs cells are inoculated to a 12-hole cell culture plate for culture, and adipogenic differentiation is induced when the cells grow to 90% fusion degree. 2) Inducing FAPs to differentiate towards the adipogenic direction for 4 days by using an adipogenic induction medium (MDI medium), then replacing the FAPs with a differentiation medium (INS medium), continuing to culture for 2 days (figure 4), setting a blank control group, culturing for 6 days by using a complete culture medium, and replacing the medium every two days; meanwhile, intramuscular precursor adipocytes obtained by the differential wall pasting method were induced to differentiate, and their polyester differentiation abilities were compared by Bodipy staining (FIG. 5).

3) Morphological changes of cells were observed and photographed at

days

2, 4, and 6 of induced differentiation (fig. 4).

4) Adopting Bodipy dyeing to detect the condition of differentiated polyester after adipogenesis induction is finished, and detecting fat synthesis and metabolism related genes by real-time fluorescence quantitative PCR (qPCR) (qPCR)PPARg, Adipoq, FABP4Etc.) (FIG. 6).

Preparing a complete culture medium: DMEM high-glucose medium (Keji organisms of Jiangsu province) was supplemented with 10% FBS (Gibco) and 1% double antibody (biologies).

MDI medium: prepared with complete medium, containing IBMX 500. mu.M, insulin 10. mu.g/mL, rosiglitazone 2. mu.g/mL and dexamethasone 1. mu.M.

INS medium: prepared with complete medium and containing 10 ug/mL insulin.

The results were:

on day 2 of differentiation, the induced group showed significant morphological changes and lipid droplets on

days

4 and 6 of differentiation (FIG. 4); bodipy staining further demonstrated that few lipid droplets appeared in the control group, whereas more lipid droplets appeared in the induced group, and that lipid droplets were significantly more in porcine FAPs cells than in intramuscular adipogenic precursor cells (fig. 5); the qPCR detection result shows that the induction group pig FAPs cellsPPARg, Adipoq, FABP4Isogene expression was significantly higher than the control group (fig. 6). The results indicate that the porcine FAPs cells can be induced and differentiated into mature adipocytes in vitro, and the porcine FAPs cells obtained by magnetic bead sorting provide a stable cell model for researching intramuscular fat deposition of pigs and production of cell culture meat.

(V) study of Gene function

Porcine FAPs cells were transfected with GFP fluorescence-tagged SV40 lentivirus and GFP fluorescence was detected 3 days after transfection (fig. 7).

The results were:

the efficiency of transfecting the porcine FAPs cells by SV40 lentivirus is high (figure 7), and the cells have normal morphology and activity after infecting lentivirus, which proves that the porcine FAPs cells can be used for gene function research in vitro.

Finally, it is also noted that the above-mentioned lists merely illustrate a few specific embodiments of the invention. It will be clear that the invention is not limited to the above examples, but that many variations are possible, such as for drug screening and preparation. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.

Claims

1. A method for separating and identifying porcine FAPs cells is characterized by comprising the following steps: the method comprises the following steps:

1) isolation and digestion of the longissimus dorsi of pigs: taking the longissimus dorsi of a 3-5-day-old pig in a sterile superclean workbench, fully shearing tissues, and then, according to the tissue volume: digestive enzyme volume = 1: 3 adding 0.2% collagenase I, digesting for 1 h at 37 ℃, filtering by a filter screen with 70 μm and a filter screen with 40 μm in sequence, centrifuging and collecting cells;

2) sorting pig FAPs cells: resuspending the cells obtained in step 1) with PBS, and comparing the volume of the cell suspension: 1 × erythrocyte lysate = 1: 9 adding erythrocyte lysate, placing at 4 ℃ for lysis for 5 min, and then centrifuging for 5 min at 400 g; resuspend the cells with 1mL of 1 XBinding buffer, add 100. mu.L dead cell remover and incubate at room temperature for 15 min, then centrifuge at 400 g for 5 min; resuspend cells at 1 × Binding buffer, check cell density with cell counter, as 10 × ⁶ Adding 2.5 μ g PDGFR alpha-Biotin antibody into each cell, incubating at 4 deg.C for 5 min, and centrifuging at 400 g for 4 min; 80 μ L of 1 Xbinding buffer resuspended cells, 20 μ L of Anti-Biotin Microbeads were added, incubated at 4 ℃ for 15 min, and centrifuged at 400 g for 4 min; resuspending the cells by 200 mu L of 1 × Binding buffer, passing through a magnetic column, wherein the cells staying on the adsorption column are FAPs cells of the pig, and eluting and collecting the cells;

3) and identifying the porcine FAPs cells to obtain a specific PDGFR alpha antibody for identifying the porcine FAPs cells by immunofluorescence.

2. The method of claim 1, further comprising: step 3) in the identification of the porcine FAPs cells, the PDGFR alpha antibody is adopted for immunofluorescence identification, and the cells have good proliferation and adipogenic differentiation potential in vitro and intramuscular fat precursor cells with remarkably high polyester differentiation capacity.

3. Use of porcine FAPs cells obtained according to the method of claim 1, characterized in that:

1) the separated pig FAPs are used for researching the formation regulation and the molecular mechanism of the pig intramuscular fat; or

2) The method is used for researching the expression rule and gene function of lipid metabolism related genes in the process of cell adipogenesis of the FAPs; or

3) Used as seed cells for cell culture meat production and used for intramuscular fat production in cell culture meat.

4. Use according to claim 3, characterized in that:

the SV40 lentivirus with GFP fluorescence label is used for transfecting pig FAPs cells, GFP fluorescence is detected after transfection for 72 hours, and the detection method is used for detecting the function and action mechanism of exogenous genes in regulation of FAPs and intramuscular adipogenesis.

5. Use according to claim 3, characterized in that: inoculating the FAPs obtained by separation into a 12-hole cell culture plate for culture, inducing FAPs to undergo adipogenic differentiation for 4 days by using an adipogenic induction culture medium MDI medium, then replacing a differentiation culture medium INS medium, continuing to culture for 2 days, and detecting the condition of FAPs differentiated polyester by adopting Bodipy staining; detection of genes associated with fat synthesis and metabolism by real-time fluorescent quantitative PCRPPARg, Adipoq, FABP4For detecting the expression of a lipid metabolism gene.

6. Use according to claim 5, characterized in that: step 4) method for inducing FAPs cell adipogenic differentiation: the MDI medium comprises IBMX 500 mu M, insulin 10 mu g/mL, rosiglitazone 2 mu g/mL and dexamethasone 1 mu M; INS medium contains insulin 10. mu.g/mL.