Method for establishing pig skeletal muscle satellite cell and pig kidney fibroblast co-culture system

Abstract

The invention discloses a method for establishing a co-culture system of porcine skeletal muscle satellite cells (PSCs) and porcine kidney fibroblasts (PKFs). The method comprises the following steps of: performing in-vitro induction on the PKFs to transdifferentiate into adipocytes, optimizing a process of the method, establishing an optimal PSCs and PKFs co-culture induced differentiation method, and selecting a co-culture proportion. According to the method for in-vitro induction of PKFs transdifferentiation and the co-culture system of PKFs and PSCs established by the invention, the PKFs can replace porcine precursor adipocytes (ADSCs) to serve as a source of seed cells in artificial meat fat, and the period of adipogenesis induced differentiation is shortened; the established PKFs and PSCs co-culture system provides a new two-dimensional model for the artificial snowflake meat; a powerful in-vitro model is provided for deep understanding of intercellular interaction and endocrine and paracrine functions of the skeletal muscle which is a highly heterogeneous tissue.

Description

Technical Field

[0001] The invention relates to the biological field, and in particular to a method for establishing a co-culture system of pig skeletal muscle satellite cells PSCs and pig kidney fibroblasts PKFs. Background Art

[0002] Artificial meat (also known as cell-cultured meat or cultivated meat) is produced on the basis of tissue engineering and the growth and repair mechanism of animal muscle, using related technologies such as cell co-culture. It has great potential for producing efficient and green meat in vitro. Therefore, more and more research is focusing on cell-cultured meat technology.

[0003] At present, most studies use a single seed cell selection, mainly muscle cells. For example, in 2021, Furuhashi M et al. used bovine muscle cells as seed cells and proposed a method for culturing 3D bovine muscle tissue in which muscle cells can be arranged along the long axis, differentiated into myotubes and contracted under electrical stimulation; in 2022, Chen Yu et al. used PSCs to explore its differentiation effect in three-dimensional hydrogels and detected the amino acid content of muscle tissue cultured in vitro; in 2019, MacQueen LA et al. cultured bovine aortic smooth muscle cells and rabbit skeletal muscle myoblasts in gelatin fiber scaffolds for the production of artificial meat. A piece of real meat has a certain tenderness and juiciness, and a single type of seed cell for artificial meat is not suitable for the production of real artificial meat. In 2022, Shahin-Shamsabadi A et al. produced mixed muscle and fat meat by adding mouse adipocytes (3T3-L1) and skeletal muscle cells (C2C12) through self-assembly layer-by-layer biomanufacturing technology, but because commercial mouse cell lines were used, large-scale production was not possible.

[0004] Well-functioning seed cells are the key to producing and cultivating fat. In 2022, Song W et al. isolated ADSCs and optimized the culture conditions of ADSCs on microcarriers (MCs) for artificial meat production. However, this type of cell has disadvantages such as cumbersome separation process and decreased cell subculture ability after cell activation; and in the process of inducing differentiation, the traditional induction method is called the "cocktail" induction method. This method has the disadvantages of a long induction cycle of adipogenic differentiation, IBMX in the culture medium, and steroids are harmful to the human body during food processing.

[0005] In 2014, Hongbo Liu et al. used PKFs for the production of cloned pigs. The results showed that PKFs showed high proliferation ability and clone-like morphology, and could be used for somatic cell nuclear transplantation (SCNT); in 2012, Richter A et al. used a culture medium with a stable karyotype to culture PKFs. The results showed that PKFs could be maintained for up to 71 passages and had a high proliferation rate and transfection efficiency; in 2023, Pasitka L et al. selected primary chicken embryonic fibroblasts (CEFs) as artificial meat seed cells, and achieved spontaneous cell immortalization, serum-free large-scale culture and artificial chicken production. PKFs, as potential seed cells for artificial meat, can replace ADSCs as a source of artificial pork fat cells, but methods for inducing PKFs to become adipogenic have not yet been reported.

[0006] Therefore, an in vitro method to induce PKFs transdifferentiation and a co-culture system of PKFs and PSCs are particularly urgent. Summary of the invention

[0007] The purpose of the present invention is to establish a method flow for inducing the transdifferentiation of pig kidney fibroblasts PKFs into adipocytes in vitro, screen the most suitable cell co-culture differentiation medium and select the most suitable cell co-culture ratio, and provide a co-culture system of pig skeletal muscle satellite cells PSCs and pig kidney fibroblasts PKFs.

[0008] The technical solution adopted by the present invention is: a method for establishing a co-culture system of porcine skeletal muscle satellite cells and porcine kidney fibroblasts, comprising the following steps:

[0009] 1) After bleeding piglets aged 1-3 days, clean and disinfect the skin surface, take piglet kidneys to isolate PKFs cells, and take piglet muscle tissue to isolate PSCs cells;

[0010] Specifically, PSCs cells are isolated from the longissimus dorsi and leg muscles of 1-3 day old piglets. The muscle tissue is cut into pieces with surgical scissors, and 1xPBS solution is added to dilute the muscle. The suspension is centrifuged at 2000g for 5 minutes, and the supernatant is discarded. 2mg / mL type I collagenase is added to the precipitate and incubated at 37°C for about 2 hours. After digestion, the cells are transferred to DMEM supplemented with 10% FBS and filtered through 100μm, 70μm and 40μm cell filters. After differential centrifugation for 2 hours, the isolated PSCs cells can be obtained.

[0011] PKFs cells were isolated from the kidneys of 1-3 day old piglets. After removing the renal capsule, the kidney tissue was cut into erosion with scissors. After washing the erosion tissue three times with PBS, pancreatin and type I collagenase (1 mg / mL) were prepared into a mixed solution at a ratio of 1:2, and the mixed solution was mixed with the washed erosion tissue at a ratio of 1:1, and digested at 37°C 5% culture conditions for 2-3 hours. After the digested tissue was passed through 100μm and 70μm cell sieves, the isolated PKFs cells were obtained.

[0012] The isolated PKFs and PSCs cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS) for 2-4 days. When the growth density of PKFs cells reached 80%-90% and the growth density of PSCs cells reached 70%-80%, they were digested with trypsin and transferred to T25 cell culture flasks for later use.

[0013] 2) Oleic acid (OA) was prepared into a 0.7 mol / LOA stock solution with DMSO, and mixed with DMEM medium containing 10% fetal bovine serum (FBS) to prepare a PKFs transdifferentiation medium containing 25 μM to 800 μM OA;

[0014] 3) taking the PKFs cells obtained in step 1) and inoculating them into the PKFs transdifferentiation medium containing 25 μM to 800 μM OA prepared in step 2) for induction;

[0015] 4) Take the PSCs cells and PKFs cells obtained in step 1), digest them with trypsin, count the cells, and mix them according to the ratio of PSCs:PKFs cells of 9:1 to 1:9 to obtain PSCs and PKFs co-culture cells for later use.

[0016] 5) Mix OA and PA, and dilute them with DMEM culture medium containing 5% fetal bovine serum (FBS) to obtain liquid A containing 25μM~800μM OA+200μM~800μM PA; Mix OA and PA, and dilute them with DMEM culture medium containing 5% horse serum (HS) to obtain liquid B containing 25μM~800μM OA+200μM~800μM PA, and cross-culture the PSCs and PKFs co-cultured cell combination obtained in step 4).

[0017] Preferably, in step 3), palmitic acid (PA) powder is dissolved in anhydrous ethanol to prepare a 50 mM stock solution PA, and the PKFs transdifferentiation medium containing OA obtained in step 2) is diluted to prepare a PKFs transdifferentiation medium containing OA and PA; the PKFs cells obtained in step 1) are inoculated in a PKFs transdifferentiation medium containing 25 μM to 800 μM OA and 200 μM to 800 μM PA to induce differentiation, and the induction time is 2 to 6 days.

[0018] Preferably, in the method for establishing a PSCs and PKFs co-culture system of the present invention, the components of the optimal PKFs transdifferentiation medium prepared in step 3) include: 600 μM OA+200 μM PA+10% FBSDMEM.

[0019] Preferably, in the method for establishing a co-culture system of PSCs and PKFs of the present invention, the most suitable co-culture differentiation medium in step 5) includes: solution A of 600 μM OA+200 μM PA+5% FBSDMEM, solution B of 600 μM OA+200 μM PA+5% HSDMEM.

[0020] Preferably, in the method for establishing a co-culture system of PSCs and PKFs of the present invention, the optimal co-culture differentiation method in step 5) comprises: culturing 2 to 4 times according to the rule of treating with solution A for 12 hours and solution B for 1 day;

[0021] Preferably, in the method for establishing a co-culture system of PSCs and PKFs of the present invention, the optimal co-culture cell number ratio of PSCs:PKFs in step 4) is 7:3.

[0022] In the method for establishing a co-culture system of PSCs and PKFs of the present invention, the adipogenic inducing agent is OA and / or PA.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The in vitro method for inducing PKFs transdifferentiation and the co-culture system of PKFs and PSCs established by the present invention improve the defects of the prior art that there is no obvious lipid droplet production and changes in adipogenic and fibrogenic marker genes after adipogenic induction using "cocktail"; and by adding PA to the 10% DMEM culture medium containing OA, the transdifferentiation efficiency of PKFs can be further improved and the transdifferentiation time can be shortened; it is helpful for PKFs to replace porcine precursor adipocytes (ADSCs) as the source of seed cells in artificial meat fat and shorten the cycle of adipogenic induction differentiation; the established co-culture system of PKFs and PSCs provides a new two-dimensional model for artificial marbled meat; and provides a powerful in vitro model for in-depth understanding of the cell-to-cell interactions, endocrine and paracrine functions of skeletal muscle, a highly heterogeneous tissue. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a cell immunofluorescence image of pig fibroblasts induced to transdifferentiate into adipocytes using the "cocktail" adipogenic induction method in Example 1. The image was collected on the 12th day of cell differentiation.

[0026] Figure 2 It is a fluorescence quantitative graph of adipogenic marker genes and fibrogenic marker genes of fibroblasts after the "cocktail" adipogenic induction method in Example 1, and the sampling time is the 12th day of cell differentiation.

[0027] Figure 3 This is an Oil Red O staining image of pig fibroblasts induced to transdifferentiate into adipocytes by adding different concentrations of oleic acid (OA) in Example 2, and the image was collected on the 6th day of cell differentiation.

[0028] Figure 4 This is a quantitative graph of the relative area of ​​lipid droplets in Example 2 inducing the transdifferentiation of porcine fibroblasts into adipocytes by adding different concentrations of oleic acid (OA).

[0029] Figure 5 This is a fluorescence quantitative graph of adipogenic marker genes and fibrogenic marker genes in pig fibroblasts induced by adding 600 μM oleic acid (OA) in Example 2. The sampling time is the 6th day of cell differentiation.

[0030] Figure 6 This is the BODIPY staining image of fibroblast transdifferentiation into adipocytes induced by adding 600 μM oleic acid (OA) and 600 μM oleic acid (OA) + 200 μM palmitic acid (PA) in Example 3. The image was collected on the 3rd day of cell differentiation.

[0031] Figure 7 This is a quantitative graph of the transdifferentiation efficiency of fibroblasts induced to transdifferentiate into adipocytes by adding 600 μM oleic acid (OA) and 600 μM oleic acid (OA) + 200 μM palmitic acid (PA) in Example 3.

[0032] Figure 8 It is a fluorescence quantitative graph of fibroblast marker genes inducing fibroblast transdifferentiation by adding 600 μM oleic acid (OA) and 600 μM oleic acid (OA) + 200 μM palmitic acid (PA) in Example 3, and the sampling time is the third day of cell differentiation.

[0033] Fig. 9 It is a fluorescence quantitative graph of adipogenic marker genes inducing fibroblast transdifferentiation by adding 600 μM oleic acid (OA) and 600 μM oleic acid (OA) + 200 μM palmitic acid (PA) in Example 3, and the sampling time is the third day of cell differentiation.

[0034] Fig.10 It is a cell immunofluorescence image of different cell ratios of co-culture of porcine muscle satellite cells (PSCs) and porcine fibroblasts (PKFs) in Example 4, and the image was collected on the 6th day of co-culture differentiation.

[0035] Fig.11 This is a quantitative graph of the degree of myotube fusion in Example 4 when pig muscle satellite cells (PSCs) and pig fibroblasts (PKFs) are co-cultured at different cell ratios.

[0036] Fig.12 This is a quantitative graph of the relative area of ​​lipid droplets in different cell ratios of co-cultured porcine muscle satellite cells (PSCs) and porcine fibroblasts (PKFs) in Example 4. DETAILED DESCRIPTION

[0037] The experimental materials and experimental schemes used in the following examples include:

[0038] Trypsin-EDTA (0.25%) (Gibco, #25200114)

[0039] Collagenase type I (Gibco, #17018029)

[0040] DMEM medium (Gibco, #12491015)

[0041] Horse serum (HS, Gibco, #16050122)

[0042] Fetal bovine serum (FBS, Gibco, #A5670701)

[0043] PBS (Gibco, #10010023)

[0044] Double antibody (Gibco, #15070063)

[0045] Dexamethasone (DEX, MCE, #HY-14648G)

[0046] Rosiglitazone (MCE, #HY-17386)

[0047] Insulin (Insulin, MCE, #HY-P0035)

[0048] Phosphodiesterase inhibitor (IBMX, MCE, #HY-12318)

[0049] Oil Red O powder (sigma, #O0625)

[0050] 4% paraformaldehyde solution (Servicebio, #G1101-500ML)

[0051] Oleic acid (OA, sigma, #O1383)

[0052] Palmitic acid(PA,MCE,#HY-N0830)

[0053] BODIPY (Thermo Fisher, #D3922)

[0054] myosin heavy chain(MyHC,Santa Cruz,#sc-376157)

[0055] Cy3 labeled goat anti-mouse IgG (Servicebio, #GB21401)

[0056] 1. After bleeding piglets aged 1-3 days, clean and disinfect the skin surface, take the piglet kidney to isolate PKFs cells, and take muscle tissue to isolate PSCs cells: PSCs are isolated from the longissimus dorsi and leg muscles of piglets aged 1-3 days. Cut the muscle tissue into pieces with surgical scissors and add 1xPBS solution to dilute the muscle. Centrifuge the suspension at 2000g for 5 minutes and discard the supernatant. Add 2mg / mL type I collagenase to the precipitate and incubate at 37℃ for about 2 hours. After digestion, transfer the cells to DMEM supplemented with 10% FBS and filter through 100μm, 70μm and 40μm cell filters. After differential centrifugation for 2 hours, PSCs cells can be obtained.

[0057] PKFs were isolated from the kidneys of 1-3 day old piglets. After removing the renal capsule, the kidney tissue was cut with scissors until it was eroded. After washing the tissue three times with PBS, pancreatin and type I collagenase (1 mg / mL) were prepared into a mixture at a ratio of 1:2, and the mixture was mixed with the washed tissue at a ratio of 1:1, and digested at 37°C 5% culture conditions for 2-3 hours. The digested tissue was passed through 100μm and 70μm cell sieves to obtain PKFs cells.

[0058] The obtained PKFs and PSCs cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS) for 2-4 days. When the growth density of PKFs reached 80%-90% and the growth density of PSCs reached 70%-80%, they were digested with trypsin and transferred to T25 cell culture flasks for later use.

[0059] 2. Prepare 0.7 mol / LOA stock solution of oleic acid (OA) powder with DMSO, and mix it with DMEM medium containing 10% FBS to prepare PKFs transdifferentiation medium containing different concentrations of OA. The obtained transdifferentiation medium can be used to induce differentiation of PKFs cells obtained in step 1;

[0060] 3. Dissolve palmitic acid (PA) powder in anhydrous ethanol to prepare a 50 mM stock solution of PA, dilute the PKFs transdifferentiation medium containing OA obtained in step 2, and prepare a PKFs transdifferentiation medium containing OA and PA. The obtained transdifferentiation medium can also be used to induce differentiation of the PKFs cells obtained in step 1;

[0061] 4. Take the PSCs cells obtained in step 1 and the induced differentiated PKFs cells obtained in step 2 or 3, digest them with trypsin, count the cells, and inoculate and co-culture them according to different PSCs:PKFs cell number ratios.

[0062] 5. Mix OA and PA, and dilute them with DMEM culture medium containing 5% FBS to obtain solution A containing 25μM~800μM OA+200μM~800μM PA; mix OA and PA, and dilute them with DMEM culture medium containing 5% HS to obtain solution B containing 25μM~800μM OA+200μM~800μM PA, and cross-culture the PSCs and PKFs co-cultured cell combination obtained in step 4).

[0063] The technical solution of the present invention is further explained below in conjunction with examples.

[0064] Example 1: The effect of inducing fibroblasts into adipogenic differentiation by using the traditional adipogenic induction method, the "cocktail" adipogenic induction method, was poor.

[0065] The traditional adipogenic induction method, the "cocktail" adipogenic induction method, was used to induce PKFs adipogenic differentiation. The adipogenic differentiation medium prepared by the "cocktail" induction method includes: Solution A: 10% FBS + 500mL DMEM + 1% double antibody + 125μL dexamethasone (DEX) + 10μL rosiglitazone + 1mg / mL insulin (Insulin,) + 5μL phosphodiesterase inhibitor (IBMX); Solution B: 10% FBS + 500mL DMEM + 1% double antibody + 1mg / mL insulin. After the preparation is completed, PKFs are inoculated into the cell culture plate, and when the cell density grows to 80%-90%, the A solution is induced for 3 days and the B solution is induced for 1 day, and the culture is cultured for 12 days.

[0066] On the 12th day of differentiation induction, Oil Red O staining was performed. 1 g of Oil Red O powder was dissolved in 20 mL of isopropanol to prepare Oil Red O concentrate. The concentrate: H2O = 3:2 was prepared into Oil Red O dilution. After filtering with three layers of filter paper, it was prepared into Oil Red O standard solution. The PKFs after induction of transdifferentiation were washed with PBS 3 times, 5 min each time, fixed with 4% paraformaldehyde solution for 30 min, washed with PBS 3 times, 5 min each time, added with Oil Red O standard solution, stained for 15 min, washed with 60% isopropanol for 15 s, and washed with PBS 3 times, 5 min each time. The expression changes of adipogenic marker genes: PPARY, LPL, ADIPOQ; fibrogenic marker genes: Co11a1, ACTA1, VIMENTIN were detected by qRT-PCR method. There was no obvious lipid droplet production and changes in adipogenic and fibrogenic marker genes after adipogenic induction using "cocktail".

[0067] Figure 1 This is the Oil Red O staining picture after 12 days of fibroblast differentiation induced by the "cocktail" induction method in Example 1. Compared with the control group, the transdifferentiation effect of PKFs induced by the "cocktail" adipogenic induction method is poor; Figure 2 This is the fluorescence quantitative graph of the adipogenic and fibrogenic marker genes in Example 1, and there is no significant change in expression level.

[0068] Example 2: PKFs can be successfully induced to transdifferentiate into adipocytes using 10% FBSDMEM medium containing OA, that is, the medium described in step 2 of Example 1 is used to induce PKFs cells

[0069] After 1g of OA powder was prepared into 0.7mol / L stock solution with 5mL DMSO, it was diluted with 10% DMEM medium, and 1.8μL, 3.6μL, 7.2μL, 14.4μL, 28.8μL, 43.2μL, and 57.6μL of the stock solution was dissolved in 50mL 10% FEB DMEM to be diluted into 25μM, 50μM, 100μM, 200μM, 400μM, 600μM, and 800μM PKFs transdifferentiation medium containing different OA concentrations. When PKFs grew to 80-90% density, PKFs transdifferentiation medium containing different OA concentrations was added. After 6 days of differentiation induction, lipid droplets were stained using Oil Red O staining technology, and the relative area of ​​lipid droplets was quantified. After induction using PKFs transdifferentiation medium with different OA concentrations, compared with the control group, when the OA concentration in the PKFs transdifferentiation medium was 600 μM, the relative area of ​​lipid droplets was larger, indicating that the transdifferentiation effect was the best.

[0070] Figure 3 This is an Oil Red O staining effect diagram of culturing PKFs using PKFs transdifferentiation medium containing different OA concentrations in Example 2, after inducing differentiation for 6 days; Figure 4 It is a quantitative diagram of the relative area of ​​lipid droplets after PKFs are treated with PKFs transdifferentiation medium containing different OA concentrations in Example 2. When the OA concentration in the PKFs transdifferentiation medium is 600 μM, the relative area of ​​lipid droplets is larger. Figure 5 This is a fluorescence quantitative graph of adipogenic and myogenic marker genes in PKFs transdifferentiation treated with 600 μM OA transdifferentiation medium and normal DMEM medium containing 10% FBS for 3 days in Example 2. After induction with 600 μM OA transdifferentiation medium, adipogenic marker genes increased significantly, and fibrogenic marker genes decreased significantly compared with the control group.

[0071] Example 3: Adding PA to 10% DMEM medium containing OA can further improve the efficiency of PKFs transdifferentiation and shorten the transdifferentiation time

[0072] 5 mg of PA was dissolved in 390 μL of anhydrous ethanol to prepare a 50 mM stock solution of PA; 43.2 μL of the stock solution OA prepared in Example 2, 200 μL of the stock solution PA prepared in this example + 43.2 μL of the stock solution OA prepared in Example 2 were dissolved in 50 mL of 10% FBSDMEM culture medium, and diluted into a transdifferentiation medium containing 600 μM OA and a transdifferentiation medium containing 200 μM PA + 600 μM OA. After inducing PKFs transdifferentiation for 3 days, the lipid droplets were stained using the BODIPY staining technique, and the BODIPY powder was dissolved in DMSO to prepare a storage solution. PKFs were fixed with paraformaldehyde for 30 minutes, and the BODIPY storage solution was diluted with water at a ratio of 1:200. After incubating the cells at room temperature for 15 minutes, they were stained with DAPI staining solution for 10 minutes and observed with a confocal microscope. And the transdifferentiation efficiency was quantified, and the transdifferentiation efficiency was calculated as follows: the number of BODIPY-stained cells / the number of unstained cells × 100%. On the third day of differentiation induction, the expression changes of adipogenic marker genes: FABP4, LPL, PPARγ, ADIPOQ, AP2; and fibrogenic marker genes: ACTA1, VIMENTIN, Co11a1, Col3a1 were detected by qRT-PCR. After adding 200μM PA to the transdifferentiation medium containing 600μM OA, the transdifferentiation efficiency reached 94.56% after 3 days of induction of transdifferentiation PKFs, which was significantly higher than that of adding 600μM OA alone; and the adipogenic marker genes after adding 200μM PA were significantly higher than those after adding 600μM OA alone, and the fibrogenic marker genes were significantly lower than those after adding 600μM OA alone.

[0073] Figure 6 This is the BODIPY staining image of PKFs induced by adding 600μM OA transdifferentiation medium alone and 200μM PA+600μM OA transdifferentiation medium for 3 days. Adding 200μM PA+600μM OA transdifferentiation medium can significantly increase the lipid droplet area after PKFs transdifferentiation; Figure 7 The comparison of transdifferentiation efficiency of PKFs showed that the transdifferentiation efficiency induced by adding 600μM OA culture medium alone could reach 74.37%, and the transdifferentiation efficiency induced by adding 200μM PA+600μM OA culture medium could reach 94.56%. Figure 8 This is the fluorescence quantitative graph of fibroblast marker genes after PKFs-induced differentiation for 3 days. Compared with the medium with 600 μM OA alone, the 200 μM PA+600 μM OA transdifferentiation medium can significantly reduce the fibroblast marker genes. Fig. 9This is a fluorescence quantitative graph of adipogenic marker genes after PKFs-induced differentiation for 3 days. Compared with the medium with 600μM OA alone, the 200μM PA+600μM OA transdifferentiation medium can significantly increase the adipogenic marker genes.

[0074] Example 4: Exploration of the optimal co-culture differentiation conditions of PSCs and PKFs

[0075] In order to explore the optimal co-culture differentiation conditions of PSCs and PKFs, solution A: 600μM OA+200μMPA+5% FBSDMEM; solution B: 5% HSDMEM; according to the rule of treating with solution A for 12h and solution B for 1 day, cultured twice; and set 11 cell mixing ratios, namely PSCs:PKFs=10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, 0:10. The cells of the 11 co-culture ratios were differentiated to the 6th day, and the differentiation was detected. Using cell immunofluorescence technology, the induced differentiated PSCs and PKFs cells were washed 3 times with PBS, 5 minutes each time, and fixed with 4% paraformaldehyde for 30 minutes. Wash with PBS 3 times, 5 min each time, prepare 0.5% Triton-X-100 in distilled water, place on a shaker at 37°C, 40r / min for 30 min; wash with PBS solution 3 times, 5 min each time, add immunofluorescence blocking solution, place on a shaker at 37°C, 40r / min for 30 min. Aspirate the blocking solution, incubate MyHC at 4°C for 14h, and wash with PBS solution 3 times, 5 min each time. Add secondary antibody Cy3-labeled goat anti-mouse IgG under light-proof conditions, place on a shaker at 37°C, 40r / min for 30 min. Wash with PBS solution 3 times, 5 min each time, add DAPI staining solution, and stand at room temperature for 10 min. Wash with PBS solution 3 times, 5 min each time, and observe the staining under a fluorescent inverted microscope; BODIPY staining technique detects the proportion of lipid droplets; the degree of myotube fusion and the area of ​​lipid droplets are counted to determine that the optimal co-culture ratio of PSCs and PKFs is 7:3.

[0076] Fig.10 These are the cell immunofluorescence images and BODIPY dye staining images of MYHC protein in 11 different cell proportions; Fig.11 It is a quantitative diagram of the degree of myotube fusion. When the ratio of PSCs:PKFs=7:3, the degree of myotube fusion is significantly lower than that of 10:0, 9:1, and 8:2, but significantly higher than that of other ratios. Fig.12 It is a quantitative graph of the relative area of ​​lipid droplets. When the ratio of PSCs:PKFs=7:3, the relative area of ​​lipid droplets is significantly higher than that of 10:0, 9:1, and 8:2, and has no significant difference compared with the ratio of 6:4 and 5:5, but is significantly lower than other ratios.

[0077] The above is a further detailed description of the present invention, which should not be regarded as a limitation on the specific implementation of the present invention. For ordinary technicians in the technical field to which the present invention belongs, simple deduction or replacement without departing from the concept of the present invention is within the protection scope of the present invention.

Claims

1. A method for establishing a co-culture system of porcine skeletal muscle satellite cells (PSCs) and porcine kidney fibroblasts (PKFs), characterized in that: The following steps are involved: 1) PKFs cells were isolated from piglet kidneys, and PSCs cells were isolated from piglet muscle tissues; the isolated PKFs and PSCs cells were cultured in DMEM medium containing 10% FBS for 2 to 4 days; when the growth density of PKFs cells reached 80%-90% and the growth density of PSCs cells reached 70%-80%, the cells were digested with trypsin and transferred to T25 cell culture flasks for later use; 2) oleic acid OA was prepared into 0.7 mol / LOA stock solution with DMSO, and mixed with DMEM medium containing 10% FBS to prepare PKFs transdifferentiation medium containing 25 μM to 800 μM OA; 3) The PKFs cells obtained in step 1) are inoculated into the PKFs transdifferentiation medium containing 25 μM to 800 μM OA prepared in step 2) to induce differentiation; 4) After the PSCs cells and PKFs cells obtained in step 1) are digested with trypsin, the cells are counted, and the ratio of PSCs:PKFs cells is 10:0 to 1:9, and the cells are mixed to obtain PSCs and PKFs co-culture cells for later use; 5) OA and PA were mixed and combined, and diluted with a DMEM medium containing 5% FBS to obtain a solution A containing 25 μM to 800 μM OA + 200 μM to 800 μM PA; OA and PA were mixed and combined, and diluted with a DMEM medium containing 5% HS to obtain a solution B containing 25 μM to 800 μM OA + 200 μM to 800 μM PA, and the PSCs and PKFs co-cultured cell combination obtained in step 4) was cross-cultured; The process of separating PKFs cells and PSCs cells in step 1) is as follows: PSCs cells are separated from the longissimus dorsi muscle and leg muscle of 1-3 day old piglets; the muscle tissue is cut into pieces, and 1x PBS solution is added to dilute the muscle to obtain a suspension; The suspension was then centrifuged at 2000 g for 5 min, and the supernatant was discarded to obtain the precipitate; 2 mg / mL type I collagenase was added to the precipitate, and after incubation at 37°C for 2 hours, the cells obtained by incubation were transferred to DMEM supplemented with 10% FBS, and filtered through 100 μm, 70 μm and 40 μm cell filters in turn; PSCs cells were isolated by differential centrifugation for 2 hours; PKFs cells are isolated from the kidneys of 1-3 day old piglets; after removing the renal capsule, the kidney tissue is cut into an erosion state with scissors; after washing the erosion-like tissue with PBS three times, pancreatic enzyme and 1 mg / mL type I collagenase are prepared into a mixed solution in a ratio of 1:2, and the mixed solution is mixed with the washed erosion-like tissue in a ratio of 1:1, and digested at 37°C 5% culture conditions for 2-3 hours, and then passed through 100μm and 70μm cell sieves in succession to obtain separated PKFs cells.

2. The method for establishing a co-culture system of porcine skeletal muscle satellite cells and porcine kidney fibroblasts according to claim 1, characterized in that: Step 3) can also be: dissolving palmitic acid (PA) powder in anhydrous ethanol to prepare a 50mM stock solution PA, diluting the PKFs transdifferentiation medium containing OA obtained in step 2) to prepare a PKFs transdifferentiation medium containing OA and PA; taking the PKFs cells obtained in step 1) and inoculating them in a PKFs transdifferentiation medium containing 25μΜ~800μΜ OA and 200μM~800μMPA to induce differentiation, and the induction time is 2 to 6 days.

3. The method for establishing a co-culture system of porcine skeletal muscle satellite cells and porcine kidney fibroblasts according to claim 2, characterized in that: Step 3) The optimal PKFs transdifferentiation medium prepared includes: 600 μM OA+200 μM PA+10% FBSDMEM.

4. The method for establishing a co-culture system of porcine skeletal muscle satellite cells and porcine kidney fibroblasts according to claim 2, characterized in that: The most suitable co-culture differentiation medium in step 5) includes: solution A containing 600 μM OA, 200 μM PA, and 5% FBSDMEM, and solution B containing 600 μM OA, 200 μM PA, and 5% HSDMEM.

5. The method for establishing a co-culture system of porcine skeletal muscle satellite cells and porcine kidney fibroblasts according to claim 1 or 2, characterized in that: In step 4), the optimal co-culture cell ratio of PSCs:PKFs is 7:3, which can induce PSCs to form myotubes and PKFs to transdifferentiate into lipid droplets.

6. The method for establishing a co-culture system of porcine skeletal muscle satellite cells and porcine kidney fibroblasts according to claim 1 or 2, characterized in that: The optimal co-culture differentiation method in step 5) is to treat with solution A for 12 hours and solution B for 1 day, and culture for 2 to 4 times.

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