A muscle stem cell active factor and its application

Abstract

The present invention relates to the field of stem cell technology, and to a muscle stem cell active factor and its application. The preparation method of the muscle stem cell active factor comprises the step of culturing using an induction medium. The present invention uses an induction medium, and the yield of the active factor of the obtained muscle stem cells is improved, which can significantly promote the proliferation of human skin fibroblasts and has excellent skin repair ability. At the same time, it can efficiently inhibit Propionibacterium acnes and has good acne removal ability. Therefore, it can be used for the preparation and production of preparations containing cell active factors, and expands new application scenarios for muscle stem cells.

Description

Technical Field

[0001] The present invention relates to the field of stem cell technology, and in particular to a muscle stem cell active factor and application thereof. Background Art

[0002] Active factors, also known as growth factors, active growth factors, and active cytokines, are a class of polypeptide substances that regulate cell growth and other cell functions by binding to specific, high-affinity cell membrane receptors. They are a combination of multiple amino acids that have the function of regulating cell growth and differentiation, also known as "peptides". Peptides are precise protein fragments, so active factors are also called active protein factors. They are distributed in various tissues of the human body, mainly in platelets and various adult and embryonic tissues and most cultured cells. The content is extremely small, but it is very important for the regulation of human cells. From the perspective of molecular structure, cell active factors are all small molecule polypeptides, most of which are composed of about 100 amino acids. Active factors have certain specificity for different types of cells. Usually, the growth of cultured cells requires the coordinated action of multiple growth factors in sequence, and tumor cells have the characteristics of autonomous growth that does not rely on growth factors.

[0003] In terms of secretion characteristics, growth factors are mainly autocrine and paracrine. Many growth factors have been purified and their structural composition has been determined. Active factors can effectively interact with skin cells, promote the nutritional metabolism of epithelial cells, prevent the skin from being damaged by ultraviolet rays, free radicals, etc., and promote the proliferation of collagen cells in the dermis. It can accelerate the repair of skin after surgery, smooth fine lines, and delay skin aging.

[0004] The active factors produced by muscle stem cells include at least the following four categories: (1) Common cell growth factors. Currently known factors include fibroblast growth factor (FGF), insulin-like growth factor-1 (IGF-1), nerve growth factor (NGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), epidermal growth factor (EGF), hepatocyte growth factor (HGF), anti-inflammatory interleukin growth factor (IL), etc. (2) Factors specifically required for muscle cell and muscle fiber growth. Myofibroblast growth factor, also known as myocyte factor (MGF), is a cytokine that has the activity of stimulating myofibroblast growth. Myocyte growth factor (MGF) is a polypeptide substance that regulates cell growth and other cell functions by binding to specific, high-affinity cell membrane receptors. (3) Anti-aging and anti-inflammatory active proteins, currently known key ones include Sirt1, Notch, Klotho, progranulin, etc. These active proteins are closely related to promoting cell growth and proliferation, wound healing and preventing long-term inflammation. (4) Factors contained in exosomes with repair functions. Muscle stem cells can secrete a large number of exosomes to act on other cells. Exosomes contain various proteins, peptides, nucleic acids and other active factors. Based on this, muscle stem cell active factors may be used to repair various muscles, including the repair and protection of women's private parts, such as repairing postpartum uterine, vaginal, and pelvic floor muscle injuries, promoting blood circulation and regenerating muscles, so as to effectively improve the relaxation of the vaginal muscles of the subjects, activate the tension of the vaginal sphincter, and then enhance the contraction function of the female vaginal muscles and vagina, improve the relaxation state of the vagina, thereby improving the tightness of the female vagina, which not only improves the contraction force of the female vagina, but also greatly improves the quality of sexual life.

[0005] Prior art CN111202749 A discloses a method for preparing a stem cell active factor composition with muscle cell repair function, first obtaining autologous or allogeneic human muscle stem cells with active regeneration ability, amplifying them in large quantities through in vitro culture, and establishing a cell line for long-term application. The cells are ultrasonically broken under low temperature conditions, the supernatant is collected after centrifugation, filtered and concentrated to obtain a muscle stem cell extract, which is combined with an extract of a stem cell culture fluid, and an active stabilizer and a preservative are added, and then mixed with a permeabilizer to obtain an active factor composition. The stem cell active factor composition can repair and regenerate damaged muscles and skin well; it can promote the improvement of skin aging.

[0006] Prior art CN116536255 B discloses a method for efficiently inducing muscle stem cells to obtain muscle fibers or skeletal muscles. The method first uses suspension culture and three muscle stem cell induction culture media for step-by-step induction, then uses a culture medium containing bovine serum and basic fibroblast growth factor for amplification, and then uses a differentiation culture medium containing specific chemical small molecules for induction of differentiation. This method can quickly induce pluripotent stem cells to obtain muscle stem cells and mature muscle fibers or skeletal muscles.

[0007] Prior art CN112553147 A discloses a cytokine combination of LR3-IGF-1, PDGF-BB, bFGF and EGF, and uses the cytokine combination for muscle stem cell culture, which can enable muscle stem cells to proliferate efficiently, greatly shorten the cell culture time, and reduce the amount of FBS used, providing a sufficient source of cells for the research of cell culture meat industry seed cells and muscle stem cells.

[0008] Prior art CN110917217 B discloses the use of muscle stem cells in the preparation of anti-inflammatory drugs. The muscle stem cells or their supernatant of the present invention are mediated by anti-inflammatory macrophages to shape their anti-inflammatory properties during the maturation of macrophages, and can effectively treat inflammatory diseases in which inflammatory macrophages are the main pathogenic cell population.

[0009] The above prior art does not mention how to mass produce it and use it in acne-removing products.

[0010] Due to the defects of existing technologies, the cultivation of muscle stem cells and new application scenarios need urgent improvement and expansion. Summary of the invention

[0011] The present invention first provides a muscle stem cell active factor, and the preparation method of the muscle stem cell active factor comprises the following steps:

[0012] (1) Obtaining muscle tissue;

[0013] (2) enzymatically hydrolyzing muscle tissue to obtain enzymatic hydrolysate;

[0014] (3) Centrifuge the enzymatic hydrolysis solution, collect the cell precipitate, and retain the muscle stem cell cluster at the bottom;

[0015] (4) Resuspend the muscle stem cell pellet from step 3, wash thoroughly, filter, and collect single cells;

[0016] (5) culturing the muscle stem cells obtained in step 4;

[0017] (6) Passaging the muscle stem cells from step 5;

[0018] (7) Cultivate the muscle stem cells from step 6 to the third generation using induction medium and collect the supernatant;

[0019] (8) The supernatant collected in step 7 is centrifuged, filtered, concentrated, and freeze-dried to obtain freeze-dried powder and muscle stem cell active factors.

[0020] In certain embodiments, the muscle stem cell activation factors include NGF, BDNF, VEGF, EGF, PDGF, HGF, and IL-6.

[0021] In certain embodiments, the enzymatic hydrolysis uses one or more of type I collagenase, type III collagenase mixture and neutral protease.

[0022] In certain embodiments, the enzymatic hydrolysis uses a mixture of type I collagenase, type III collagenase and neutral protease.

[0023] In certain embodiments, the induction medium is DMEM medium supplemented with stem cell growth factor, glutathione and long-chain recombinant human insulin-like growth factor.

[0024] In certain embodiments, the long-chain recombinant human insulin-like growth factor is LR3-IGF-I.

[0025] In certain embodiments, the concentrations of the stem cell growth factor, glutathione and long-chain recombinant human insulin-like growth factor are 0.1-1 ng / ml, 0.1-0.5 mg / ml and 20-100 ng / ml, respectively.

[0026] In certain embodiments, the concentrations of the stem cell growth factor, glutathione and long-chain recombinant human insulin-like growth factor are 0.5 ng / ml, 0.2 mg / ml and 50 ng / ml, respectively.

[0027] The present invention also provides an application of the muscle stem cell active factor, which is an application in preparing a preparation containing the cell active factor. The preparation may include an anti-acne preparation, such as a facial mask.

[0028] Finally, the present invention provides a muscle stem cell active factor induction culture medium, wherein the induction culture medium is prepared by adding stem cell growth factor, glutathione and long-chain recombinant human insulin-like growth factor into DMEM culture medium.

[0029] Compared with the prior art, the present invention has at least the following beneficial effects:

[0030] (1) The present invention uses an induction culture medium, and the yield of active factors of the obtained muscle stem cells is improved. The active factors of the muscle stem cells prepared contain higher levels of NGF, BDNF, VEGF, EGF, PDGF, HGF, IL-6 and other factors;

[0031] (2) The active factors of muscle stem cells prepared in the present application can significantly promote the proliferation of human skin fibroblasts and have excellent skin repair ability. At the same time, they can effectively inhibit Propionibacterium acnes and have good acne removal ability. Therefore, they can be used in the preparation and production of facial masks, which expands new application scenarios for muscle stem cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 .Comparison of proliferation rates. DETAILED DESCRIPTION

[0033] In order to make the technical problems, technical solutions and advantages to be solved by the present invention more clear, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0034] Example 1 Preparation of muscle stem cell active factors

[0035] A method for preparing muscle stem cell active factors, comprising the following steps:

[0036] (1) Under sterile conditions, obtain 5 g of human muscle tissue. Rinse the muscle tissue with physiological saline and crush it until the particle size of the tissue is no larger than 1 mm to obtain muscle tissue homogenate, which is then dispensed into 50 ml centrifuge tubes at a rate of 5 ml per tube.

[0037] (2) Add equal volumes of type I collagenase, type III collagenase mixture and neutral protease to the centrifuge tube in step (1), digest at 200R for 60 min in a constant temperature air bath shaker at 37°C, and then inactivate the enzyme to obtain muscle tissue enzymatic hydrolysate.

[0038] (3) Centrifuge the resulting enzymatic hydrolysate at 2000 rpm for 10 minutes, collect the cell precipitate, and retain the muscle stem cell cluster at the bottom.

[0039] (4) Add 10 ml of physiological saline to the centrifuge tube containing the muscle stem cell cluster, repeatedly blow and resuspend the cells to make a uniform single-cell suspension, wash thoroughly, let the cells pass through a filter with a diameter of 70 microns, collect single cells, and then centrifuge at 2000 rpm for 5 minutes, discard the upper layer of liquid, and retain the muscle stem cell cluster at the bottom; repeat this washing step twice.

[0040] (5) After thorough washing, add an appropriate amount of physiological saline and repeatedly blow the cells to make a single-cell suspension of muscle stem cells. Add the suspension to a culture bottle containing DMEM complete medium and place it in a 37°C incubator with a 5% CO2 concentration for culture.

[0041] (6) When the cells are in culture on the 3rd and 6th days, the medium should be changed in time. On the 7th to 10th day, when 3 to 5 cell colonies reach more than 80%, the cells can be subcultured.

[0042] (7) Remove the culture supernatant; add buffer to rinse the culture flask, remove the supernatant and add trypsin to digest for 3-5 minutes. After digestion, observe the cells under a microscope and tap the culture flask with your palm to ensure that the cells are completely detached from the bottom wall of the cell flask.

[0043] (8) Add culture medium to stop digestion and blow off the cells, transfer to a new centrifuge tube, add buffer to rinse the culture bottle, collect the remaining cells and centrifuge at 400g×3min. After centrifugation, discard the supernatant, add culture medium to resuspend and blow the cells evenly, take 100μL of cell suspension for cell counting and viability detection, and culture cells at a ratio of 1:3. Take the third generation muscle stem cells for the experiment.

[0044] (9) Cultivate muscle stem cells to the third generation according to the steps of step (8), discard the supernatant; add 10 ml of induction medium, culture at 5% CO2 concentration and 37°C for 2-3 days to induce muscle stem cells to produce active factors, and collect the supernatant after culture. The induction medium is DMEM medium with stem cell growth factor (SCF), glutathione and long-chain recombinant human insulin-like growth factor (LR 3 -IGF-Ⅰ), the concentrations of which were 0.5ng / ml, 0.2mg / ml, and 50ng / ml, respectively.

[0045] (10) The collected supernatant is centrifuged, filtered using a 0.22 μm rapid filter, and then transferred into a concentrator for concentration, followed by freeze-drying to obtain a freeze-dried powder; thus, the muscle stem cell active factor is obtained.

[0046] (11) Store the lyophilized powder from step (10) in a -20°C refrigerator for later use.

[0047] Comparative Example 1

[0048] Except that the induction medium only contained SCF and glutathione, other conditions were the same as those in Example 1.

[0049] Comparative Example 2

[0050] Except that the induction medium contains only SCF and LR 3Except for IGF-Ⅰ, other conditions were the same as those in Example 1.

[0051] Comparative Example 3

[0052] Except that the induction medium contains only glutathione and LR 3 Except for IGF-Ⅰ, other conditions were the same as those in Example 1.

[0053] Comparative Example 4

[0054] Except that the induction medium contained only SCF, other conditions were the same as those in Example 1.

[0055] Comparative Example 5

[0056] Except that the induction medium contains only glutathione, other conditions are the same as those in Example 1.

[0057] Comparative Example 6

[0058] Except that the induction medium contains only LR 3 Except for IGF-Ⅰ, other conditions were the same as those in Example 1.

[0059] Comparative Example 7

[0060] Except that the induction medium does not contain SCF, glutathione, LR 3 Except for IGF-Ⅰ, other conditions were the same as those in Example 1.

[0061] Example 2 Active Factor Detection

[0062] For the detection of muscle stem cell active factors in Example 1 and Comparative Examples 1 to Comparative Examples 7, Elisa was mainly used to detect factors such as NGF, BDNF, VEGF, EGF, PDGF, HGF, IL-6, and the relative expression levels of each group were calculated based on the expression levels of Comparative Example 7. The results are shown in Table 1. The relative expression levels of each active factor in Example 1 were significantly improved, indicating that the induction medium had a significant effect and that each component achieved a synergistic effect.

[0063] Table 1 Relative expression of each active factor

[0064] Experimental Group NGF BDNF VEGF EGF PDGF HGF IL-6 Example 1 6.4 7.3 10.2 7.4 6.6 7.7 11.2 Comparative Example 1 3.0 2.8 3 2.9 4.1 6.0 4.7 Comparative Example 2 3.3 3.1 4 3.2 4.5 6.8 5.7 Comparative Example 3 3.2 3.5 5 3.1 4.2 6.7 6.3 Comparative Example 4 1.6 1.4 1.6 1.5 1.9 2.3 1.8 Comparative Example 5 1.5 1.6 1.8 1.9 2.2 2.4 1.5 Comparative Example 6 1.8 1.6 2.3 2.2 2.6 3.2 2.3

[0065] Example 3 Skin repair effect test

[0066] Human skin fibroblasts HSF in the logarithmic growth phase were taken and inoculated on a 96-well plate at 5×105 / mL per well. Five replicate wells were set up in each group. After the cells adhered to the wall, DMEM complete medium and CCK-8 solution were mixed in a volume ratio of 100:1 and the cells were incubated for 2 hours. The supernatant was aspirated and the absorbance at a wavelength of 450nm was detected with an enzyme marker. The cells in the 96-well plate were washed once with PBS. The experimental groups were cultured with DMEM culture medium containing muscle stem cell activity factors according to Example 1 and Comparative Examples 1 to Comparative Examples 7. The content of muscle stem cell activity factors in all experimental groups was the same, all of which were 20 μg / mL. The control group: only DMEM complete culture medium was added for culture; the absorbance values ​​at a wavelength of 450nm were measured for 96 hours, and the results were as follows Figure 1 As shown, it is shown that the muscle stem cell active factor of Example 1 has the highest effect in promoting the proliferation of human skin fibroblasts.

[0067] Example 4 In vitro antibacterial test of Propionibacterium acnes

[0068] The muscle stem cell active factors of Example 1 and Comparative Examples 2-7 were prepared into active factor solutions with different gradient concentrations. 1 mL of the above active factors was taken and 0.1 mL of 5×10 7 CFU / mL of activated Propionibacterium acnes suspension. A control tube with no active factor was set up. After anaerobic culture at 37℃ for 48h, the results were observed and the MIC (minimum inhibitory concentration) of each sample was determined. The test was repeated 3 times for each sample in each group. The results are shown in Table 2.

[0069] Table 2 Results of in vitro antibacterial test of Propionibacterium acnes

[0070] Experimental Group MIC mg / ml Example 1 0.15625 Comparative Example 1 0.625 Comparative Example 2 0.3125 Comparative Example 3 0.3125 Comparative Example 4 1.25 Comparative Example 5 1.25 Comparative Example 6 0.625 Comparative Example 7 5.00

[0071] It can be seen from Table 2 that the muscle stem cell active factor of Example 1 of the present invention has the best antibacterial effect on Propionibacterium acnes.

[0072] In summary, the muscle stem cell active factor of the present application has a high content of each effective ingredient, and it has the strongest human skin fibroblast proliferation ability, which means it can promote skin repair. At the same time, it can also effectively inhibit Propionibacterium acnes and has good acne removal ability. It can be well applied to acne removal preparations such as facial masks.

[0073] The above is 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 principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a muscle stem cell active factor, characterized in that: The preparation method comprises the following steps: (1) Obtaining muscle tissue; (2) Enzymatically hydrolyzing muscle tissue to obtain enzymatic hydrolysate; (3) Centrifuge the enzymatic hydrolysis solution, collect the cell precipitate, and retain the muscle stem cell cluster at the bottom; (4) Resuspend the muscle stem cell pellet from step 3, wash thoroughly, filter, and collect single cells; (5) culturing the muscle stem cells obtained in step 4; (6) Passaging the muscle stem cells from step 5; (7) Cultivate the muscle stem cells from step 6 to the third generation, use induction medium, and collect the supernatant; (8) Centrifuging, filtering, concentrating, and freeze-drying the supernatant collected in step 7 to obtain freeze-dried powder and muscle stem cell active factors; The muscle stem cell active factors include NGF, BDNF, VEGF, EGF, PDGF, HGF and IL-6; The enzymatic hydrolysis uses a mixed solution of type I collagenase, type III collagenase and a neutral protease; The induction medium is a DMEM medium in which stem cell growth factor, glutathione and long-chain recombinant human insulin-like growth factor are added; The long-chain recombinant human insulin-like growth factor is LR3-IGF-Ⅰ; The concentrations of the stem cell growth factor, glutathione and long-chain recombinant human insulin-like growth factor are 0.5 ng / ml, 0.2 mg / ml and 50 ng / ml respectively.

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