Application of dihydroflavonoid compound naringin in promoting proliferation and differentiation of porcine muscle stem cells

Abstract

The invention provides an application of dihydroflavonoid compound naringin in promoting proliferation and differentiation of porcine muscle stem cells to form myotubes, which is characterized in that the naringin is added into a DMEM / F12 culture medium containing 15% of fetal calf serum and 1% of double antibodies and a DMEM differential culture medium containing 2% of horse serum and 1% of double antibodies until the final concentration is 70 mu M, and the porcine muscle stem cells are respectively treated in proliferation and differentiation stages, hoechst 33342 staining and immunofluorescence staining methods are used for respectively judging the number of cells in the proliferation stage and the fluorescence area, fluorescence intensity and nuclear fusion rate of Myosin in myotubes formed in the differentiation stage. The relative proliferation multiple of the muscle stem cells treated by adding naringin and the fluorescence area, the fluorescence intensity and the nuclear fusion rate of Myosin in myotubes are obviously higher than those of a blank control group. By adopting the method, proliferation and differentiation of the porcine muscle stem cells can be promoted, and a new thought is provided for maintaining cell dryness and large-scale production of cultured meat.

Description

Technical Field

[0001] The invention relates to the field of cell cultured meat, and in particular to the application of a dihydroflavonoid compound naringin in promoting the proliferation and differentiation of pig muscle stem cells. Background Art

[0002] Meat has always been regarded as an excellent source of protein and is highly favored by consumers. However, with the development of the economy and the rapid growth of the global population, the demand for protein has risen sharply. Although traditional animal husbandry can meet part of the demand, the environmental problems it brings, such as ecological damage and waste of water resources, have made it difficult to maintain sustainability, resulting in a huge gap in protein supply. Cultured meat, as a revolutionary future food production technology, is not only expected to fill this gap, but also to significantly reduce the harm caused by traditional animal husbandry, bringing a greener and more sustainable solution to future food production.

[0003] As the most promising cultured meat seed cells, muscle stem cells have the function of repairing damaged muscle tissue, and can effectively differentiate into myotubes, and finally generate muscle fibers, expressing muscle-specific myosin heavy chains. However, muscle stem cells face the problem of decreased proliferation and differentiation ability during in vitro culture. Studies have shown that the proportion of PAX7, an important marker of muscle stem cells, will decrease from 100% to 30% to 50% after the muscle stem cells are just isolated and passaged for 3 times, indicating that it is very difficult to maintain the proliferation and differentiation ability of muscle stem cells under intensive and large-scale expansion conditions in vitro, which is very unfavorable for the continuous culture of cells in vitro.

[0004] At present, many substances with antioxidant properties have been proven to effectively protect cells under oxidative stress environments such as H2O2. They regulate cell damage responses through specific signaling pathways and significantly reduce cell apoptosis. This discovery not only reveals the great potential of antioxidant substances in cell protection, but also provides a new research direction for cultured meat.

[0005] Naringin is a natural flavonoid compound that is widely found in the peel and pulp of citrus fruits. It is easy to extract and separate. It is not only highly safe, but also has cardiovascular protection, anti-inflammatory, anti-atherosclerotic and anti-oxidative stress capabilities. However, there are few reports on the effect of naringin on the in vitro culture of muscle stem cells. Summary of the invention

[0006] Based on the above problems existing in the prior art, the present invention designs and develops a high-content screening method for functional factors that promote the proliferation and differentiation of pig muscle stem cells.

[0007] The technical solution of the present invention is:

[0008] Application of naringin in promoting proliferation of muscle stem cells and / or promoting myogenic differentiation of muscle stem cells in vitro culture

[0009] Furthermore, the muscle stem cells are porcine muscle stem cells.

[0010] Furthermore, the muscle stem cells are pig muscle stem cells no later than P5 generation.

[0011] Furthermore, naringin was added to the proliferation medium and differentiation medium during the in vitro proliferation culture and in vitro differentiation induction stages of muscle stem cells, respectively.

[0012] Furthermore, the proliferation medium is a basal medium added with recombinant human fibroblast growth factor (bFGF), and the volume ratio of the recombinant human fibroblast growth factor to the basal medium is 1:2000; the basal medium is a DMEM / F12 medium added with serum and 100X penicillin-streptomycin double antibody, and the volume ratio of DMEM / F12 to serum and double antibody is 84%:15%:1%.

[0013] Furthermore, the differentiation medium is prepared by adding horse serum and double antibodies to DMEM high-glucose solution, and the volume ratio of DMEM high-glucose solution to horse serum and double antibodies is 97%:2%:1%.

[0014] Furthermore, in the in vitro proliferation culture of muscle stem cells, the final concentration of naringin is 70 μM to 90 μM, preferably, the final concentration of naringin is 90 μM.

[0015] Furthermore, the final concentration of naringin in the in vitro induced differentiation of muscle stem cells was 70 μM.

[0016] Furthermore, the in vitro proliferation culture of muscle stem cells includes the following steps: adding a proliferation medium containing naringin to a cell culture dish to revive pig muscle stem cells at a concentration of 1×10 5 The cells were transferred into a large dish and cultured in a 37°C, 5% CO2 incubator. The medium was changed every two days and the cells were counted after three days of culture.

[0017] Furthermore, the in vitro differentiation of muscle stem cells includes the following steps: pig muscle stem cells are cultured at 8×10 4 The cells were inoculated into a cell culture dish pre-coated with matrix gel, and the proliferation of porcine muscle stem cells was continuously cultured with proliferation medium. The medium was changed every two days until the cell confluence reached more than 90% under a microscope. The original proliferation medium was removed, the culture dish was washed with PBS, and differentiation medium containing naringin was added to induce cell differentiation. Half of the medium was changed every two days to induce differentiation in vitro.

[0018] In a particular embodiment, since naringin is not completely soluble in water, it can be pre-dissolved in an appropriate solvent and then added to the proliferation medium or differentiation medium. For example, dimethyl sulfoxide (DMSO) can be used to dissolve it into a high-concentration mother solution and then added to the corresponding culture medium. To prevent DMSO from causing cytotoxicity, the ratio of the mother solution volume to the proliferation medium volume is recommended to be less than or equal to 1:2000 when adding the sample, and the mother solution is sealed with aluminum foil and stored at -80°C away from light.

[0019] The structural formula of naringin described in the present invention is:

[0020] The beneficial effects of the present invention are

[0021] 1. The present invention successfully promotes the proliferation of pig muscle stem cells through the above method, and induces cells to form myotubes in the differentiation stage, significantly improving differentiation indicators such as nuclear fusion rate.

[0022] 2. Compared with the blank treatment group without adding naringin, the proliferation multiples of cells treated with the present invention increased by 134%-171%.

[0023] 3. Immunofluorescence showed that the number of marker protein Myosin in the myotubes obtained by the method of the present invention was significantly higher than that in the control group, and the nuclear fusion rate in the differentiation stage was 50.43%, which was 1.19 times higher than that in the control group.

[0024] 4. In view of the problems of muscle stem cells' proliferation and differentiation ability decline during in vitro culture, which is not conducive to large-scale culture, the present invention can significantly promote the proliferation and differentiation of pig muscle stem cells cultured in vitro. Its application in promoting the proliferation and differentiation of pig muscle stem cells can fill the gap in research, provide new ideas for the large-scale culture of cell-cultured meat, and contribute to the large-scale production of cell-cultured meat. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The Hoechst 33342 staining images of proliferation for 72 hours were collected under a fluorescent inverted microscope, where A, B, and C are blank control groups, and D, E, and F are naringin 70μM treatment group, 80μM treatment group, and 90μM treatment group, respectively.

[0026] Figure 2 is the relative proliferation rate after naringin treatment for 72 hours.

[0027] Figure 3 This is a confocal immunofluorescence image acquired 5 days after differentiation, in which the green part is myosin, the blue part is the cell nucleus, and the treatment group numbering sequence is the same as above.

[0028] Figure 4 It is a bar graph of the results of differentiation after naringin treatment, where A is the fluorescence area, B is the fluorescence intensity, and C is the nuclear fusion rate. DETAILED DESCRIPTION

[0029] The present invention is further explained below with reference to the examples, but the examples do not limit the present invention in any form.

[0030] Example 1

[0031] 1 Materials and methods

[0032] 1.1 Reagents used in the experiment

[0033] Australian fetal bovine serum, donor horse serum, DMEM / F12 nutrient medium, DMEM high glucose solution (purchased from GIBCO, USA, catalog number: C11995500BT), bFGF, phosphate buffered saline (PBS), 100X penicillin-streptomycin double antibody, 0.25% trypsin; DMSO; Hoechst 33342 staining solution, Myosin primary antibody, mouse secondary antibody, 4% paraformaldehyde, Triton, bovine serum albumin solution (BSA), matrix gel (purchased from Nanjing Weiwo Biotechnology Co., Ltd., catalog number: V900933-100G), naringin (purchased from Shanghai Yuanye Biotechnology Co., Ltd.).

[0034] 1.2 Experimental methods

[0035] 1.2.1 Sample configuration

[0036] Preparation of basal culture medium: Add fetal bovine serum and double antibody to DMEM / F12 culture medium, the volume ratio of DMEM / F12 to fetal bovine serum and double antibody is 84%:15%:1%, and put the prepared basal culture medium into a -20℃ refrigerator for standby use; Preparation of cell freezing solution: Add DMSO to fetal bovine serum to make the DMSO concentration 10%, and put the prepared cell freezing solution into a 4℃ refrigerator for standby use;

[0037] Preparation of proliferation medium: Add recombinant human fibroblast growth factor (bFGF) to the basal medium.

[0038] Preparation of differentiation medium: Use DMEM high-glucose solution as the main culture medium, add low-concentration horse serum and double antibody at a ratio of 97%:2%:1%, mix well and place the differentiation medium in a -20℃ refrigerator for later use;

[0039] Preparation of naringin mother solution: dissolve naringin in DMSO to prepare a mother solution, then take an appropriate amount of the mother solution and dilute it with the basal culture medium according to the concentration gradient. The final ratio of DMSO to basal culture medium is not greater than 1:2000.

[0040] 1.2.2 In vitro proliferation and culture of porcine muscle stem cells

[0041] According to 1.5×10 5 The cells were inoculated into a 10 cm cell culture dish pre-coated with rat tail collagen. Recombinant human fibroblast growth factor (bFGF) was added to the basal culture medium, and the two were mixed at a ratio of 2000:1 to prepare a proliferation medium. The naringin mother solution was diluted to 70, 80, and 90 μM using the proliferation medium, and the cells were cultured using it. The medium was changed every two days, and the original culture medium was removed on the third day and the counting results were recorded.

[0042] 1.2.3 Induced differentiation of porcine muscle stem cells

[0043] Resuscitated P4 porcine muscle stem cells were cultured in a 10 cm dish in a 37°C, 5% CO2 incubator and passaged 3 days later at a rate of 8 × 10 4 The cells were inoculated into a 3.5 cm cell culture dish pre-coated with matrix gel, and porcine muscle stem cells were continuously cultured with proliferation medium. The medium was changed every two days until the cell confluence reached more than 90% under a microscope. The original proliferation medium was removed, and the culture dish was washed with 1 mL of PBS. 2 mL of differentiation medium containing naringin was added to induce cell differentiation. Half of the medium was changed every two days, that is, half of the original differentiation medium was removed and an equal volume of fresh differentiation medium was added. The differentiation was terminated after five days.

[0044] 1.2.4 Detection of Proliferation of Pig Muscle Stem Cells

[0045] After 72 hours of in vitro proliferation culture, the culture medium was removed and DMEM / F12 culture medium containing Hoechst 33342 staining solution (ratio 1:2000) was added and protected from light. The large dish with staining solution was wrapped in tin foil and placed in a 37°C incubator for 10 minutes and then taken out and images were acquired using a fluorescent inverted microscope.

[0046] 1.2.5 Detection of pig muscle stem cell differentiation

[0047] After 5 days of in vitro differentiation, perform immunofluorescence experiments, remove the culture medium, and wash twice with PBS. 3 min / time. Add 4% paraformaldehyde (diluted with 1X PBS) and fix at room temperature for 20-30 min or overnight at 4 degrees. Wash 3 times with PBS, 5 min / time. Aspirate PBS. Add 0.5% Triton X-100 (diluted with 1X PBS) and permeabilize at room temperature for 15-20 min. Wash 3 times with PBS shaker, 5 min / time. Aspirate PBS. Prepare 5% BSA or 10% goat serum and block for 1 hour. Incubate primary antibody: add enough diluted primary antibody to each glass bottom culture dish, put it in a wet box, and incubate overnight at 4°C; incubate secondary antibody: recover primary antibody, wash 3 times with PBS, 5 min / time. Aspirate PBS. Add diluted fluorescent secondary antibody, incubate in a wet box at 20-37°C for 1 hour, and immerse twice with PBS, 5 min each time. DAPI or Hochest was added and incubated for 5 min (protected from light), the sample was stained for nucleus, and then washed twice with PBS, each time for 5 min. The images were observed and collected under a confocal microscope or fluorescence microscope.

[0048] 1.2.6 Statistical methods

[0049] As a preferred method, SPSS27.0 statistical software was used to perform a normality test on the data, and one-way analysis of variance was used for comparison among multiple groups. The analysis method was LSD, and P < 0.05 was considered to be significantly different.

[0050] 2 Experimental results

[0051] 2.1 Experimental results of muscle stem cell proliferation stage

[0052] 2.1.1 Image acquisition results of cell proliferation after three days of naringin treatment

[0053] The results of DAPI staining images of cell proliferation treated with functional factor naringin are as follows Figure 1 As shown, the results showed that the number of cells in the groups treated with naringin at three concentration gradients of 70, 80, and 90 μM was greater than that in the control group, and the number of cells increased with the increase of concentration.

[0054] 2.1.2 Analysis of cell proliferation results after three days of naringin treatment

[0055] The relative proliferation multiples were obtained by SPSS 27.0 statistical software. Figure 2 The results showed that the addition of naringin significantly promoted cell proliferation. The 90μM naringin treatment group had the best effect, with a relative proliferation multiple of 1.71 times. The 70μM naringin treatment group had a poor effect, but the relative proliferation multiple still reached 1.34 times.

[0056] 2.2 Experimental results of muscle stem cell differentiation stage

[0057] 2.2.1 Image acquisition results of cells treated with naringin for five days

[0058] The collection results of cell differentiation images treated with the functional factor naringin are as follows Figure 3 As shown, the results showed that compared with the blank control group, the protein area of ​​the naringin treatment group at three concentration gradients of 70, 80, and 90 μM was larger, and the number of cell nuclei in the protein was more.

[0059] 2.2.2 Analysis of the results of cell differentiation after five days of naringin treatment

[0060] The results of the analysis using SPSS 27.0 statistical software are as follows: Figure 4 The results showed that the nuclear fusion rate of the 70μM naringin treatment group was significantly higher than that of the control group, while the 80 and 90μM treatment groups failed to significantly promote cell differentiation.

[0061] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of naringin in promoting the proliferation of muscle stem cells and / or promoting the myogenic differentiation of muscle stem cells in vitro culture.

2. The use according to claim 1, characterized in that: The muscle stem cells are pig muscle stem cells.

3. The use according to claim 1, characterized in that: The muscle stem cells are pig muscle stem cells no later than P5.

4. The use according to claim 1, characterized in that: Naringin was added to the proliferation medium and differentiation medium during the in vitro proliferation culture and in vitro differentiation induction stages of muscle stem cells, respectively.

5. The use according to claim 4, characterized in that: The proliferation medium is a basal medium added with recombinant human fibroblast growth factor (bFGF), and the volume ratio of the recombinant human fibroblast growth factor to the basal medium is 1:2000; the basal medium is a DMEM / F12 medium added with serum and 100X penicillin-streptomycin double antibody, and the volume ratio of DMEM / F12 to serum and double antibody is 84%:15%:1%.

6. The use according to claim 4, characterized in that: The differentiation medium is prepared by adding horse serum and double antibodies to a DMEM high-glucose solution, and the volume ratio of the DMEM high-glucose solution to the horse serum and double antibodies is 97%:2%:1%.

7. The use according to claim 5, characterized in that: The final concentration of naringin is 70 μM to 90 μM, preferably, the final concentration of naringin is 90 μM.

8. The use according to claim 6, characterized in that: The final concentration of naringin was 70 μM.

9. The use according to claim 4, characterized in that: The in vitro proliferation culture of muscle stem cells includes the following steps: adding a proliferation medium containing naringin to a cell culture dish to revive pig muscle stem cells at a rate of 1×10 5 The cells were transferred into a large dish and cultured in a 37°C, 5% CO2 incubator. The medium was changed every two days and the cells were counted after three days of culture.

10. The use according to claim 4, characterized in that: In vitro differentiation of muscle stem cells includes the following steps: Porcine muscle stem cells were cultured at 8×10 4 The cells were inoculated into a cell culture dish pre-coated with matrix gel, and the proliferation of porcine muscle stem cells was continuously cultured with proliferation medium. The medium was changed every two days until the cell confluence reached more than 90% under a microscope. The original proliferation medium was removed, the culture dish was washed with PBS, and differentiation medium containing naringin was added to induce cell differentiation. Half of the medium was changed every two days to induce differentiation in vitro.

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