A stem cell composition for promoting skin cell repair and its preparation method and application

Abstract

The present invention discloses a stem cell composition for promoting skin cell repair, and a preparation method and application thereof, and belongs to the technical field of skin repair. The present invention is the first to mix the skin repair peptide isolated from farmed giant salamanders with the supernatant of umbilical cord mesenchymal stem cells to prepare a stem cell composition for promoting skin cell repair. The skin repair peptide and the supernatant of umbilical cord mesenchymal stem cells in the composition provided by the present invention have a synergistic effect, and the combination of the two can effectively increase the proliferation of fibroblasts, promote the secretion of type I and type III collagen, and is beneficial to the repair of skin damage, and has good commercial application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of skin repair, and in particular to a stem cell composition for promoting skin cell repair, and a preparation method and application thereof. Background Art

[0002] Skin repair refers to repairing the skin barrier, which is the first line of defense for the skin to deal with the outside world. A healthy skin barrier can resist external pollutants, such as dust, ultraviolet rays, bacteria and other external stimuli, avoid excessive water loss, and keep the skin smooth and moisturized. When the skin barrier is damaged, there will be varying degrees of redness, peeling, dryness, tightness and other symptoms. At this time, the damaged skin barrier needs to be repaired.

[0003] Usually, minor damage to the skin barrier can be repaired by using skin care products and improving lifestyle habits. For example, basic moisturizing, avoiding ultraviolet radiation, eating more fruits and vegetables, and insisting on going to bed early and getting up early can improve the skin condition. If the damage is slightly more serious, drugs can be used to repair the skin barrier. Commonly used drugs include vitamin E cream, polysulfonated mucopolysaccharide cream, and retinoic acid cream. These ointments have a good moisturizing effect on the skin. If the skin damage is caused by allergic factors, glucocorticoid ointments should be used. It should be noted that long-term use of glucocorticoid ointments may cause a certain dependence on the drug, and it will also make the skin barrier very fragile, so it should be used under the guidance of a doctor.

[0004] At present, studies have confirmed that mesenchymal stem cells can be used to treat soft tissue filling and regeneration, hair regeneration, scar reduction and anti-aging. Mesenchymal stem cells can improve skin health by increasing skin thickness, collagen formation and reducing wrinkles. However, skin repair products based on mesenchymal stem cells have not yet been launched. How to effectively improve and enhance the skin repair function of mesenchymal stem cells is currently a hot research and development topic in the industry. Summary of the invention

[0005] In view of the technical problems existing in the prior art, the present invention provides a stem cell composition for promoting skin cell repair, and a preparation method and application thereof. The present invention is the first to mix the skin repair peptide separated from farmed giant salamanders with the supernatant of umbilical cord mesenchymal stem cells to prepare a stem cell composition for promoting skin cell repair. In the composition, the skin repair peptide and the supernatant of umbilical cord mesenchymal stem cells have a synergistic effect. The combination of the two can effectively increase the proliferation of fibroblasts, promote the secretion of type I and type III collagen, and is beneficial to the repair of skin damage, and has good commercial application prospects.

[0006] One of the purposes of the present invention is to provide a stem cell composition for promoting skin cell repair, which comprises 10-20 mL of umbilical cord mesenchymal stem cell supernatant and 3-8 mg of skin repair peptide.

[0007] Preferably, the stem cell composition for promoting skin cell repair comprises 10 mL of umbilical cord mesenchymal stem cell supernatant and 3 mg of skin repair peptide.

[0008] Preferably, the stem cell composition for promoting skin cell repair comprises 15 mL of umbilical cord mesenchymal stem cell supernatant and 5 mg of skin repair peptide.

[0009] Preferably, the stem cell composition for promoting skin cell repair comprises 20 mL of umbilical cord mesenchymal stem cell supernatant and 8 mg of skin repair peptide.

[0010] Preferably, the amino acid sequence of the skin repair peptide is as shown in SEQ ID NO.1.

[0011] Further preferably, the method for preparing the umbilical cord mesenchymal stem cell supernatant comprises the following steps:

[0012] 1) Under sterile conditions, take fresh umbilical cord, wash it thoroughly with sterile PBS, remove the umbilical artery and umbilical vein, peel off the outer membrane, keep only the Wharton's jelly, cut it into small pieces for later use;

[0013] 2) Digest with 100-200U hyaluronidase and 50-100U protease in sequence at 37°C for 30-45 minutes; filter the digestion solution with a 100-200 mesh filter, centrifuge the collected filtrate at 1000g for 10-20 minutes, discard the supernatant, and wash with low-glucose DMEM medium for 3-5 times to obtain umbilical cord mesenchymal stem cells;

[0014] 3) Take 2×10 5 -5×10 6 Umbilical cord mesenchymal stem cells were inoculated into 25 mL culture bottles for subculture and cultured continuously for 3-5 days;

[0015] 4) After the culture is completed, the supernatant of the umbilical cord mesenchymal stem cells after passage in step 3) is collected by centrifugation at 1200g and 4°C for 10-20 min, and filtered through a 0.22 μm filter membrane for later use;

[0016] 5) Centrifuge the supernatant of umbilical cord mesenchymal stem cells in step 4) at 12000g and 4°C for 30-45 min, aspirate the supernatant to remove cell impurities and precipitate; filter with a 0.22 μm filter membrane and set aside.

[0017] Further preferably, the skin repair peptide is isolated from freshly cultured giant salamanders and obtained by enzymatic hydrolysis and high performance liquid chromatography identification.

[0018] Preferably, the separation method of the skin repair peptide comprises the following steps:

[0019] 1) Separate the muscle tissue of freshly cultured giant salamander, grind it into a homogenate, add 100-200U trypsin and 80-150U neutral protease, enzymatically hydrolyze it at 37℃ for 10-16 hours, centrifuge at 12000rpm for 30-45min, and take the supernatant;

[0020] 2) Use Unique AutoPrep high pressure preparative liquid chromatography for separation and purification, collect the eluate, and perform quantitative analysis.

[0021] Another object of the present invention is to provide a method for preparing a stem cell composition for promoting skin cell repair, characterized in that the method comprises directly dissolving the above-mentioned skin repair peptide in the supernatant of umbilical cord mesenchymal stem cells, and then stirring and clarifying it.

[0022] One of the purposes of the present invention is to provide a use of a stem cell composition for promoting skin cell repair in the preparation of a product for promoting skin cell repair.

[0023] Preferably, the use is by applying the stem cell composition directly to the damaged skin surface.

[0024] The advantages of the present invention are as follows: The present invention provides a stem cell composition for promoting skin cell repair prepared by mixing skin repair peptides isolated from cultured giant salamanders with umbilical cord mesenchymal stem cell supernatant for the first time. In the composition, the skin repair peptides and the umbilical cord mesenchymal stem cell supernatant have a synergistic effect, and the combination of the two can effectively increase fibroblast proliferation, promote type I and type III collagen secretion, and is beneficial to skin damage repair, and has good commercial application prospects. DETAILED DESCRIPTION

[0025] The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand the present invention more clearly.

[0026] The following embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Based on the specific embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without creative work belong to the protection scope of the present invention.

[0027] In the examples of the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art; in the examples of the present invention, unless otherwise specified, the technical means used are conventional means well known to those skilled in the art. Example 1

[0028] A stem cell composition for promoting skin cell repair, comprising 10 mL of umbilical cord mesenchymal stem cell supernatant and 3 mg of skin repair peptide, wherein the amino acid sequence of the skin repair peptide is shown in SEQ ID NO.1.

[0029] The method for preparing the umbilical cord mesenchymal stem cell supernatant comprises the following steps:

[0030] 1) Under sterile conditions, take a fresh umbilical cord, wash it thoroughly with sterile PBS, remove the umbilical artery and umbilical vein, peel off the outer membrane, keep only the Wharton's jelly, cut it into small pieces, and set aside;

[0031] 2) Digest with 100U hyaluronidase and 50U protease in sequence at 37°C for 30 minutes; filter the digestion solution with a 100-mesh filter, centrifuge the collected filtrate at 1000g for 10 minutes, discard the supernatant, and wash with low-glucose DMEM medium for 3 times to obtain umbilical cord mesenchymal stem cells;

[0032] 3) Take 2×10 5 Umbilical cord mesenchymal stem cells were inoculated into 25 mL culture bottles for subculture and cultured for 3 consecutive days;

[0033] 4) After the culture is completed, the supernatant of the umbilical cord mesenchymal stem cells after passage in step 3) is collected by centrifugation at 1200g and 4°C for 10 min, and filtered through a 0.22 μm filter membrane for later use;

[0034] 5) Centrifuge the supernatant of umbilical cord mesenchymal stem cells in step 4) at 12000g and 4°C for 30 min, aspirate the supernatant to remove cell impurities and precipitate; filter with a 0.22 μm filter membrane and set aside.

[0035] The skin repair peptide is separated from freshly cultured giant salamanders and obtained by enzymatic hydrolysis and high-performance liquid chromatography identification. The specific method steps include:

[0036] The muscle tissue of freshly cultured giant salamander was separated and ground into a homogenate, and 100U of trypsin and 80U of neutral protease were added for enzymatic hydrolysis at 37°C for 10 hours, and the mixture was centrifuged at 12000rpm for 30 minutes, and the supernatant was collected;

[0037] 2) Use Unique AutoPrep high pressure preparative liquid chromatography for separation and purification, collect the eluate, and perform quantitative analysis. Example 2

[0038] A stem cell composition for promoting skin cell repair, comprising 15 mL of umbilical cord mesenchymal stem cell supernatant and 5 mg of skin repair peptide, wherein the amino acid sequence of the skin repair peptide is shown in SEQ ID NO.1. Example 3

[0039] A stem cell composition for promoting skin cell repair, comprising 20 mL of umbilical cord mesenchymal stem cell supernatant and 8 mg of skin repair peptide, wherein the amino acid sequence of the skin repair peptide is shown in SEQ ID NO.1.

[0040] Comparative Example 1

[0041] A stem cell composition for promoting skin cell repair, comprising 20 mL of umbilical cord mesenchymal stem cell supernatant.

[0042] Comparative Example 2

[0043] A stem cell composition for promoting skin cell repair comprises 8 mg of skin repair peptide dissolved in 20 mL of 0.9% saline.

[0044] Test Example 1

[0045] Effect of stem cell composition on fibroblast proliferation: The MTT cell proliferation detection kit (Cat. No. 11465007001, purchased from Roche) was used to detect the effect of the stem cell composition on the proliferation of fibroblast HFF-1. The specific detection steps can be adjusted according to the instructions of the kit. The blank group is DMEM complete medium, and the amount of HFF-1 cells in the experimental group and the control group is 1×10 5 The specific results are shown in Table 1.

[0046] In Table 1, experimental groups 1-3 correspond to the stem cell compositions prepared in Examples 1-3, respectively; and control groups 1-2 correspond to the stem cell compositions prepared in Examples 1-2, respectively.

[0047] Table 1 Analysis of fibroblast proliferation

[0048]

[0049] The test results are shown in Table 1: The stem cell compositions obtained by any of the experimental groups 1-3 of the present invention can effectively promote the proliferation of fibroblasts, while the proliferation promoting effect is not significant when using comparative examples 1 and 2 alone. The reason is that the supernatant of umbilical cord mesenchymal stem cells is rich in a variety of small molecule short peptides and other nutrients, which synergistically promote the proliferation of fibroblasts with the skin repair peptides from giant salamanders, and have a good skin repair effect.

[0050] Test Example 2

[0051] Effect of stem cell composition on collagen secretion of fibroblasts: The fibroblasts HFF-1 obtained from different treatment groups were cultured for 24 h, the cells were collected, and the secretion of type I and type III collagen was detected by ELISA kit (Cat. No. EH7533 / EH2866, purchased from Wuhan Fine Biotechnology Co., Ltd.). The results are shown in Table 2.

[0052] In Table 2, experimental groups 1-3 correspond to the stem cell compositions prepared in Examples 1-3, respectively; and control groups 1-2 correspond to the stem cell compositions prepared in Examples 1-2, respectively.

[0053] Table 2 Results of type I and type III collagen secretion

[0054]

[0055] From the results in Table 2, it can be seen that the stem cell composition of the present invention can efficiently promote the secretion of type I and type III collagen by fibroblasts, and the effect is significantly better than that of the control group (P < 0.05), while comparative examples 1 and 2 also partially promote the secretion of type I and type III collagen by fibroblasts, but the effect is not obvious. When the two are used in combination, the stem cell composition has a synergistic effect on the secretion of collagen by fibroblasts, further proving that the stem cell composition of the present invention has good skin repair function.

[0056] The above embodiments are intended to illustrate the essential content of the present invention, but are not intended to limit the protection scope of the present invention. Those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and protection scope of the technical solution of the present invention.

Claims

1. A stem cell composition for promoting skin cell repair, characterized in that: The composition consists of 10-20 mL of umbilical cord mesenchymal stem cell supernatant and 3-8 mg of skin repair peptide; the amino acid sequence of the skin repair peptide is shown in SEQ ID NO.

1.

2. The stem cell composition according to claim 1, wherein The composition consists of 10 mL of umbilical cord mesenchymal stem cell supernatant and 3 mg of skin repair peptide.

3. The stem cell composition according to claim 1, wherein The composition consists of 15 mL of umbilical cord mesenchymal stem cell supernatant and 5 mg of skin repair peptide.

4. The stem cell composition according to claim 1, wherein The composition consists of 20 mL of umbilical cord mesenchymal stem cell supernatant and 8 mg of skin repair peptide.

5. The stem cell composition according to any one of claims 1 to 4, characterized in that: The method for preparing the umbilical cord mesenchymal stem cell supernatant comprises the following steps: 1) Under sterile conditions, take a fresh umbilical cord, wash it thoroughly with sterile PBS, remove the umbilical artery and umbilical vein, peel off the outer membrane, keep only the Wharton's jelly, cut it into small pieces, and set aside; 2) Digest with 100-200U hyaluronidase and 50-100U protease in sequence at 37°C for 30-45 minutes; filter the digestion solution with a 100-200 mesh filter, centrifuge the collected filtrate at 1000g for 10-20 minutes, discard the supernatant, and wash with low-glucose DMEM medium for 3-5 times to obtain umbilical cord mesenchymal stem cells; 3) Take 2×10 5 -5×10 6 Umbilical cord mesenchymal stem cells were inoculated into 25 mL culture bottles for subculture and cultured continuously for 3-5 days; 4) After the culture is completed, the supernatant of the umbilical cord mesenchymal stem cells after passage in step 3) is collected by centrifugation at 1200g and 4°C for 10-20 min, and filtered through a 0.22 μm filter membrane for later use; 5) Centrifuge the supernatant of umbilical cord mesenchymal stem cells in step 4) at 12000g and 4°C for 30-45 min, aspirate the supernatant to remove cell impurities and precipitate; filter with a 0.22 μm filter membrane and set aside.

6. The method for preparing the stem cell composition according to any one of claims 1 to 5, characterized in that: The method comprises directly dissolving the skin repair peptide in the supernatant of umbilical cord mesenchymal stem cells, and stirring and clarifying the supernatant.

7. Use of the stem cell composition according to any one of claims 1 to 5 in the preparation of a product for promoting skin cell repair.

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