Skin repairing liquid containing stem cell extract and preparation method thereof

Abstract

The invention provides a skin repairing liquid containing stem cell extract and a preparation method thereof, and belongs to the technical field of biology. According to the invention, adipose-derived mesenchymal stem cells are used as starting cells, and main secretory active peptides are prepared by means of enzymolysis and the like, and are used for constructing the skin repair liquid disclosed by the invention. Results show that the specific stem cell secreted active peptide has the function of promoting skin epidermal cell proliferation, effectively promotes wound healing, can effectively inhibit infection of common pathogenic bacteria, has an antibacterial function while repairing skin, and has a good market application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a skin repair liquid containing stem cell extract and a preparation method thereof. Background Art

[0002] A skin repair liquid is a liquid product used for repairing and protecting the skin, and is usually used for treating various skin problems, such as wounds, burns, acne, skin healing after surgery, and skin aging, dryness and water shortage.

[0003] However, the existing skin repair liquid products have a single function, and some patients may have allergic reactions after use, and they cannot take into account both skin repair and antibacterial functions. Especially when it comes to patients with open wounds or extensive burns, multiple skin repair products need to be comprehensively used to effectively achieve wound healing and skin infection treatment, which will inevitably increase the burden on patients and bring adverse treatment and intervention experiences. Summary of the Invention

[0004] In view of the technical problems existing in the prior art, the present invention provides a skin repair liquid containing stem cell extract and a preparation method thereof. The present invention discovers that specific stem cell-secreted active peptides have the function of promoting the proliferation of skin epidermal cells, effectively promoting wound healing, and at the same time can effectively inhibit the infection of common pathogenic bacteria, having an antibacterial function while repairing the skin, and having good market application prospects.

[0005] Specifically, the present invention first provides a skin repair liquid, which is composed of 5-10 parts of adipose mesenchymal stem cell-secreted active peptide, 10-15 parts of glycerol, 20-25 parts of hyaluronic acid, and 50-65 parts of deionized water.

[0006] Preferably, the adipose mesenchymal stem cell-secreted active peptide is selected from any one or a combination of several of SEQ ID NO.1-2.

[0007] Preferably, a skin repair liquid containing stem cell extract is composed of 5 parts of adipose mesenchymal stem cell-secreted active peptide, 10 parts of glycerol, 20 parts of hyaluronic acid, and 50 parts of deionized water.

[0008] More preferably, the adipose mesenchymal stem cell-secreted active peptide is selected from SEQ ID NO.1.

[0009] Preferably, a skin repair liquid containing stem cell extract is composed of 10 parts of adipose mesenchymal stem cell-secreted active peptide, 15 parts of glycerol, 25 parts of hyaluronic acid, and 65 parts of deionized water.

[0010] Preferably, the adipose mesenchymal stem cell-secreted active peptide is selected from SEQ ID NO.2.

[0011] The present invention also provides a method for preparing a skin repair liquid, and the method comprises the following steps:

[0012] 1) In deionized water, glycerol and hyaluronic acid are sequentially added, and continuous stirring is carried out to fully dissolve them;

[0013] 2) Active peptides secreted by adipose mesenchymal stem cells are sequentially added to step 1) and fully dissolved in a water bath environment at 25°C to obtain the product.

[0014] Preferably, the method for preparing the skin repair liquid comprises the following steps:

[0015] 1) In 65 parts of deionized water, 10 parts of glycerol and 20 parts of hyaluronic acid are sequentially added, and continuous stirring is carried out to fully dissolve them;

[0016] 2) 5 parts of active peptides secreted by adipose mesenchymal stem cells are sequentially added to step 1) and fully dissolved in a water bath environment at 25°C to obtain the product.

[0017] Preferably, the method for preparing the skin repair liquid comprises the following steps:

[0018] 1) In 50 parts of deionized water, 15 parts of glycerol and 25 parts of hyaluronic acid are sequentially added, and continuous stirring is carried out to fully dissolve them;

[0019] 2) 10 parts of active peptides secreted by adipose mesenchymal stem cells are sequentially added to step 1) and fully dissolved in a water bath environment at 25°C to obtain the product.

[0020] Further preferably, the present invention also provides the use of the active peptides secreted by adipose mesenchymal stem cells in the preparation of skin repair products.

[0021] Further preferably, the skin repair method includes promoting the proliferation of skin fibroblasts and / or inhibiting pathogen infection.

[0022] Further preferably, the pathogen is one or more selected from Escherichia coli, Staphylococcus aureus, Streptococcus, and Malassezia.

[0023] Further preferably, the product further includes common excipients in cosmetics.

[0024] The advantages of the present invention are as follows: The present invention uses adipose mesenchymal stem cells as starting cells, and prepares the main secreted active peptides through enzymatic hydrolysis and other means, and constructs the skin repair liquid of the present invention with these peptides. The results show that the specific stem cell-secreted active peptides have the function of promoting the proliferation of skin epidermal cells, effectively promoting wound healing, and at the same time can effectively inhibit the infection of common pathogenic bacteria, having an antibacterial function while repairing the skin, and having good market application prospects. Detailed Embodiments

[0025] The following combines specific embodiments to further elaborate on the present invention, 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 those of ordinary skill in the art without creative efforts fall within the protection scope of the present invention.

[0027] In the embodiments 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 embodiments of the present invention, unless specifically specified, the technical means used are all conventional means well-known to those skilled in the art.

[0028] Example 1

[0029] Extraction method of active peptides secreted by adipose mesenchymal stem cells, the method comprising the following steps:

[0030] 1) Take adipose tissue for trypsin digestion to prepare a single-cell suspension, and use a flow cytometer to sort adipose mesenchymal stem cells of + CD44 + CD63 + CD105 + CD14 - CD31 - CD45 - CD144 - ;

[0031] 2) Place the adipose mesenchymal stem cells obtained in step 1) in a 6-well plate containing K-SFM medium for continuous passage culture;

[0032] 3) When the cell density reaches ≥80%, remove the medium and wash with 1×PBS;

[0033] 4) Add 250 μL of RIPA lysis buffer per well to step 3), gently pipette for 3 s, centrifuge at 10,000 g at 4 °C for 5 min, and take the supernatant for standby;

[0034] 5) Add collagenase to the supernatant of step 4) for enzymatic hydrolysis treatment at 37 °C for 2 h;

[0035] 6) Filtration and concentration: Filter the enzymatic hydrolysis supernatant of step 5) using an ultrafiltration membrane, and retain polypeptide molecules with a molecular weight cut-off of 5500 - 8000 Da for standby;

[0036] 7) Active peptide identification: Take the concentrated solution of step 6) for HPLC and Obitrap Fusion Lumos mass spectrometer sequencing identification, and collect the active peptide samples for standby;

[0037] 8) Vacuum drying: Vacuum concentrate the active peptides identified in step 7) to a concentration of 18 °Bé, and spray dry them into fine powder for standby.

[0038] After HPLC and mass spectrometer identification and analysis, a total of two main intracellular active peptides were obtained, and their sequences are shown as SEQ ID NO.1 - 2 in sequence.

[0039] Example 2

[0040] A skin repair solution, characterized in that the skin repair solution is composed of 5 parts of adipose mesenchymal stem cell - secreted active peptides, 10 parts of glycerol, 20 parts of hyaluronic acid, and 65 parts of deionized water prepared by the above method.

[0041] Among them, the sequence of the adipose mesenchymal stem cell - secreted active peptides is as shown in SEQ ID NO.1.

[0042] The preparation method of the skin repair solution includes the following steps:

[0043] 1) Take 65 parts of deionized water, and sequentially add 10 parts of glycerol and 20 parts of hyaluronic acid, and continuously stir to fully dissolve them;

[0044] 2) Sequentially add 5 parts of adipose mesenchymal stem cell - secreted active peptides to step 1) and dissolve them fully in a water bath environment at 25 °C to obtain the product.

[0045] Example 3

[0046] A skin repair solution, characterized in that the skin repair solution is composed of 10 parts of adipose mesenchymal stem cell - secreted active peptides, 15 parts of glycerol, 25 parts of hyaluronic acid, and 50 parts of deionized water prepared by the above method.

[0047] Among them, the sequence of the adipose mesenchymal stem cell - secreted active peptides is as shown in SEQ ID NO.2.

[0048] The preparation method is the same as that of Example 2.

[0049] Control

[0050] The preparation method steps of Comparative Example 1 are the same as those of Example 2, except that the active ingredient of the skin repair liquid does not contain the active peptide secreted by adipose mesenchymal stem cells, but uses an equal amount of deionized water as a supplement. The preparation method is the same as that of Example 2.

[0051] Test Example 1

[0052] Effect of the skin repair liquid on the proliferation of fibroblasts: The MTT cell proliferation detection kit (Model 20311, purchased from Shanghai Shangbao Biotechnology Co., Ltd.) was used to detect the proliferation effect of the above-mentioned examples and comparative examples on NHDF normal human skin fibroblasts (Product number ZB063, purchased from Shanghai Yaji Biotechnology Co., Ltd.). The specific detection steps can be routinely adjusted according to the kit instructions. Among them, the blank group is DMEM complete medium, Experimental Groups 1-2 respectively correspond to DMEM complete medium containing the skin repair liquid prepared in Examples 2-3, and the control group contains DMEM complete medium with the skin repair liquid prepared in the comparative example. The cell dosage in each treatment group is 1×10 4 cells.

[0053] Table 1 Analysis of fibroblast proliferation

[0054] Group OD value Blank group 0.206±0.024 Experimental group 1 0.725±0.120 Experimental group 2 0.846±0.035 Control group 0.253±0.032

[0055] The detection results are shown in Table 1: The blank group cannot promote the proliferation of fibroblasts, while the skin repair liquid obtained by any one of Experimental Groups 2-3 of the present invention can efficiently promote the proliferation of fibroblasts, and its proliferation-promoting effect is significantly better than that of the control group (P < 0.05), further confirming that the active peptide secreted by adipose mesenchymal stem cells obtained by the preparation method of the present invention has a good skin repair effect and can effectively promote skin wound healing.

[0056] Test Example 2

[0057] Analysis of the bacteriostatic performance of the skin repair liquid: The plate counting method was used to statistically analyze the bactericidal rates of the skin repair liquid of the present invention against Escherichia coli, Staphylococcus aureus, Streptococcus, and Malassezia. The results are shown in Table 2.

[0058] Table 2 Analysis of bacteriostatic performance

[0059] Group Escherichia coli (bactericidal rate%) Staphylococcus aureus (bactericidal rate%) Streptococcus (bactericidal rate%) Malassezia (bactericidal rate%) Blank group 2.35±0.05 1.25±0.04 2.05±0.08 1.21±0.01 Experimental group 1 85.35±0.25 82.25±0.15 85.05±0.12 80.21±0.24 Experimental group 2 90.25±0.14 89.61±0.20 88.55±0.35 86.24±0.42 Control group 3.15±0.04 2.32±0.04 2.46±0.03 1.57±0.05

[0060] The detection results are shown in Table 2: The skin repair liquid of the present invention has excellent bacteriostatic effects on common skin infection pathogens. It can not only effectively repair skin damage, but also inhibit the reproduction of bacteria at the skin damage site, effectively preventing skin diseases caused by pathogen infections. This further fully demonstrates that the skin repair liquid of the present invention has good repair and healing abilities.

[0061] It is necessary to point out here that the above embodiments are only for further elaboration and explanation of the technical solution of the present invention, rather than a further limitation on the technical solution of the present invention. The method of the present invention is only a preferred implementation solution and is not used to limit the protection scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A skin repair liquid, characterized in that, The skin repair liquid comprises 5 - 10 parts of adipose mesenchymal stem cell - secreted active peptides, 10 - 15 parts of glycerol, 20 - 25 parts of hyaluronic acid, and 50 - 65 parts of deionized water, wherein the adipose mesenchymal stem cell - secreted active peptides are selected from any one or several combinations of SEQ ID NO.1 - 2.

2. The skin repair liquid according to claim 1, wherein The skin repair liquid is composed of 5 parts of adipose mesenchymal stem cell - secreted active peptides, 10 parts of glycerol, 20 parts of hyaluronic acid, and 50 parts of deionized water.

3. The skin repair liquid according to claim 2, wherein The adipose mesenchymal stem cell - secreted active peptides are selected from SEQ ID NO.

1.

4. The skin repair liquid according to claim 1, characterized in that, The skin repair liquid comprises 10 parts of adipose mesenchymal stem cell - secreted active peptides, 15 parts of glycerol, 25 parts of hyaluronic acid, and 65 parts of deionized water prepared by the above - mentioned method.

5. The skin repair liquid according to claim 4, wherein, The adipose mesenchymal stem cell - secreted active peptides are selected from SEQ ID NO.

2.

6. The preparation method of the skin repair liquid according to any one of claims 1-5, characterized in that The method comprises the following steps: 1) In deionized water, glycerol and hyaluronic acid are added sequentially, and continuously stirred to make them fully dissolved. 2) The adipose mesenchymal stem cell - secreted active peptides are added sequentially to step 1), and made to fully dissolve in a water - bath environment at 25°C, thus obtaining the product.

7. The preparation method according to claim 6, characterized in that, The preparation method of the skin repair liquid comprises the following steps: 1) In 65 parts of deionized water, 10 parts of glycerol and 20 parts of hyaluronic acid are added sequentially, and continuously stirred to make them fully dissolved. 2) 5 parts of adipose mesenchymal stem cell - secreted active peptides are added sequentially to step 1) and made to fully dissolve in a water - bath environment at 25°C, thus obtaining the product.

8. The preparation method according to claim 7, characterized in that, The preparation method of the skin repair liquid comprises the following steps: 1) In 50 parts of deionized water, 15 parts of glycerol and 25 parts of hyaluronic acid are added sequentially, and continuously stirred to make them fully dissolved. 2) 10 parts of adipose mesenchymal stem cell - secreted active peptides are added sequentially to step 1) and made to fully dissolve in a water - bath environment at 25°C, thus obtaining the product.

9. Use of adipose mesenchymal stem cell-secreted bioactive peptides in the preparation of skin repair products, characterized in that, The adipose mesenchymal stem cell - secreted active peptides are selected from any one or several combinations of SEQ ID NO.1 - 2.