Human umbilical cord mesenchymal stem cell extract and application thereof in skin care products

Abstract

The invention discloses a human umbilical cord mesenchymal stem cell extract and application thereof in a skin care product, belongs to the technical field of biological medicine, and provides application of the human umbilical cord mesenchymal stem cell extract prepared in the first aspect in preparation of a skin care product composition for relieving and repairing skin. The soothing and repairing skin care product composition comprises the human umbilical cord mesenchymal stem cell extract provided in the first aspect and a pharmaceutically acceptable carrier, the carrier is a hydrogel preparation, and the volume ratio of the human umbilical cord mesenchymal stem cell extract to the hydrogel preparation is 1: 1. Human umbilical cord mesenchymal stem cells are extracted through in-vitro cell culture and are mixed with a gel preparation according to a certain proportion to prepare the skin care product composition, the skin care product composition is used for relieving and repairing skin, the human umbilical cord mesenchymal stem cell extract can stably exert the effect, the human umbilical cord mesenchymal stem cell extract and the tea tree essence cooperate with each other, relieving and repairing of the skin are promoted, and the skin care effect is improved. And the skin barrier is prevented from being damaged.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and particularly relates to an extract of human umbilical cord mesenchymal stem cells and its application in skin care products. Background Art

[0002] Human umbilical cord mesenchymal stem cells (MSCs) exist in neonatal umbilical cord tissue and are a type of multipotent stem cells. These stem cells have the ability of self-renewal and multi-directional differentiation potential, and can differentiate into various tissue cells. The extract of umbilical cord mesenchymal stem cells is obtained by extracting from these stem cells, and is rich in various bioactive components such as cytokines, polypeptides, small molecule proteins and amino acids.

[0003] The immunogenicity of the extract of human umbilical cord mesenchymal stem cells has fewer components compared with cells, so its immunogenicity can be further reduced, and the extract retains the main biological characteristics of human umbilical cord mesenchymal stem cells, including low immunogenicity and paracrine function. The suspension preparation of the extract of human umbilical cord mesenchymal stem cells can also stimulate the production of collagen, promote the self-repair and regeneration of the skin, thereby improving the skin quality. However, when directly coated on the skin surface, the active ingredients do not have the ability to stably retain and release. Therefore, the current suspension preparation of the extract of human umbilical cord mesenchymal stem cells often fails to achieve the expected therapeutic effect in actual clinical applications. Summary of the Invention

[0004] In view of the above situation, to overcome the defects of the prior art, the present invention provides an extract of human umbilical cord mesenchymal stem cells and its application in skin care products. The extract of human umbilical cord mesenchymal stem cells is obtained by in vitro cell culture, and is mixed with a gel preparation in a certain proportion to prepare a skin care product composition, which is used for soothing and repairing the skin, and can enable the extract of human umbilical cord mesenchymal stem cells to stably exert its efficacy, synergize with tea tree essence, promote the soothing and repair of the skin, and prevent damage to the skin barrier.

[0005] Specifically, the present invention is achieved through the following solutions:

[0006] In the first aspect, the present invention provides an extract of human umbilical cord mesenchymal stem cells, which is prepared by the following method:

[0007] Step 1: Resuscitate the human umbilical cord mesenchymal stem cells selected from the cell bank, add them to 8 ml of DMEM culture medium containing 10% fetal bovine serum, then centrifuge at 1000 r / min, with a centrifugal radius of 10 cm for 5 min, and discard the supernatant; add 2 ml of culture medium and blow evenly, transfer the cells to a culture dish, add about 5 ml of culture medium, and place them in an incubator for culture;

[0008] Step 2: After collecting the cells, wash them once with PBS and once with cell lysate. After removing the supernatant, resuspend with 1 volume of cell lysate (generally, 1 μL of the extract is obtained from 10 5 cells), and lyse on ice for 30 - 45 min;

[0009] Step 3: Disrupt the cells with an ultrasonic cell disruptor until the cell nuclei are completely disrupted. Centrifuge at 4°C and 15,000 x g for 15 min, collect the supernatant, aliquot, and store at -80°C.

[0010] Furthermore, the cells collected in Step 2 are passage 3 - 5 human umbilical cord mesenchymal stem cells.

[0011] When using flow cytometry to detect the surface antigens of the mesenchymal stem cell extract prepared above, the positive rates of surface protein expression are CD29 (98.81%); CD44 (92.10%); CD105 (98.65%). The experimental results of surface protein expression can show that the morphology of the human umbilical cord mesenchymal stem cell extract prepared by the preparation method in this application is complete, and the quality and quantity of the obtained extract are better.

[0012] In a second aspect, the present invention provides the use of the human umbilical cord mesenchymal stem cell extract prepared in the first aspect above in the preparation of a skin care product composition for soothing and repairing the skin.

[0013] Among them, the soothing and repairing skin care product composition includes the human umbilical cord mesenchymal stem cell extract proposed in the first aspect and a pharmaceutically acceptable carrier. The carrier is a hydrogel preparation, and the volume ratio of the human umbilical cord mesenchymal stem cell extract to the hydrogel preparation is 1:1.

[0014] Furthermore, the pH of the hydrogel preparation is 5.5 - 7, and every 100 ml of the hydrogel preparation includes the following components:

[0015] Tween 80 5 - 15 g;

[0016] Hyaluronic acid 0.5 - 2 ml;

[0017] Tea tree essence 2 - 8 ml;

[0018] Vitamin E 0.5 - 2 ml.

[0019] Furthermore, the soothing and repairing skin care product is used for soothing skin inflammation.

[0020] Furthermore, the soothing and repairing skin care product is used for repairing the sensitive skin barrier.

[0021] According to the above technical solutions, the beneficial effects of the present invention are as follows:

[0022] The human umbilical cord mesenchymal stem cell extract proposed in this application and its application in skin care products use Tween 80 as a dispersant and stabilizer, which can fully disperse the human umbilical cord mesenchymal stem cell extract, tea tree essence and vitamin E in the gel preparation. The human umbilical cord mesenchymal stem cell extract is rich in various growth factors, cytokines and exosomes, and is used for skin repair and anti-inflammatory in wound healing. Hyaluronic acid can promote skin moisture retention, enhance skin barrier function, and has a certain anti-inflammatory effect. The strong moisturizing performance of hyaluronic acid can combine with the active ingredients in the human umbilical cord mesenchymal stem cell extract to further improve the skin moisture and elasticity. Both of them have a certain anti-inflammatory effect at the same time, which can enhance the resistance to skin inflammation and oxidative stress. The repair and anti-inflammatory functions of the two complement the anti-inflammatory effect of the tea tree essence (EGCG component) to jointly improve the skin condition. Moreover, the antioxidant and moisturizing properties of hyaluronic acid and vitamin E act synergistically on damaged skin tissues, jointly promoting skin repair and regeneration with the human umbilical cord mesenchymal stem cell extract, maintaining the natural barrier function of the skin, reducing water loss, accelerating the growth and renewal of epidermal cells, and accelerating the wound healing process. In addition, the moisturizing ability of hyaluronic acid can be combined with the oil control effect of the tea tree essence to help maintain the water-oil balance of the skin and ensure that the active ingredients act on the microenvironment of the skin tissue. Description of the Drawings

[0023] The drawings are used to provide a further understanding of the present invention and constitute a part of the specification. They are used together with the embodiments of the present invention to explain the present invention and do not constitute a limitation to the present invention.

[0024] Figure 1 It is the detection result of inflammatory factors in D4 serum in Example 5;

[0025] Figure 2 It is the detection result of inflammatory factors in D10 serum in Example 5. Detailed Embodiments

[0026] The present invention discloses a human umbilical cord mesenchymal stem cell extract and its application. Those skilled in the art can draw on the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all regarded as included in the present invention. The methods and applications of the present invention have been described through preferred embodiments. Relevant personnel can obviously make changes or appropriate alterations and combinations to the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0027] Example 1: Preparation of human umbilical cord mesenchymal stem cell extract.

[0028] The preparation method of the human umbilical cord mesenchymal stem cell extract proposed in this example is as follows:

[0029] Step 1: Resuscitate the human umbilical cord mesenchymal stem cells selected from the cell bank, add them to 8 ml of DMEM culture medium containing 10% fetal bovine serum, then centrifuge at 1000 r / min, with a centrifugal radius of 10 cm, for 5 min, and discard the supernatant; add 2 ml of culture medium and blow evenly, transfer the cells to a culture dish, add about 5 ml of culture medium, and place them in an incubator for culture;

[0030] Step 2: After collecting the cells, wash them once with PBS and once with cell lysate. After removing the supernatant, resuspend them with 1 volume of cell lysate (generally, 1 μL of extract is obtained from 10 5 cells), and lyse them on ice for 30 - 45 min;

[0031] Step 3: Use an ultrasonic crusher to break the cells until the cell nuclei are completely broken, centrifuge at 4 °C and 15000 xg for 15 min, collect the supernatant, and aliquot and store it at -80 °C.

[0032] Cell lysate formula: 0.1 mol / L HEPES pH 8.2; 0.05 mol / L NaCl; 5 μmol / L MgCl; 1 μmol / L DTT; protease inhibitor (100x); 0.1 μmol / L PMSF; H0, add 5 μL of 0.5 mol / L EDTA to every 250 μL of lysate (use lysate without added EDTA during the washing process).

[0033] Perform fluorescence labeling and nano-flow cytometry detection on the human umbilical cord mesenchymal stem cell extract sample prepared by the above method. The antibodies for fluorescence labeling are CD29, CD44, and CD105. The detection results are shown in Table 1;

[0034] Table 1: Experimental results of surface protein expression

[0035]

[0036] Example 2: Preparation of hydrogel preparation.

[0037] The gel preparation prepared in this example, as the carrier of the human mesenchymal stem cell extract prepared in Example 1, also completely retains effective biological functions and therapeutic capabilities, and can act synergistically with the human mesenchymal stem cell extract to jointly act on soothing and repairing damaged skin.

[0038] In this example, the preparation method of the hydrogel preparation: According to the ratio, add Tween 80, tea tree essence, and vitamin E to the hyaluronic acid hydrogel, and adjust the pH of the solution to between 6.5 - 7.5 with an appropriate pH regulator.

[0039] Example 3: Preparation of soothing and repairing skin care product composition.

[0040] In this embodiment, the soothing and repairing skin care product composition includes human umbilical cord mesenchymal stem cell extract and a hydrogel preparation. The above extract and preparation are fully mixed according to a volume ratio of 1:1, and after being evenly mixed, they are sub-packed and stored refrigerated.

[0041] Example 4: In vitro antibacterial effect

[0042] Prepare the skin care product composition: According to the preparation methods of Examples 1-3 and the following formula, prepare the compositions of experimental groups 1-5 respectively, and the blank group 6 is physiological saline.

[0043] Table 2: Formulas of experimental groups

[0044]

[0045] Cell culture: From the facial skin lesion contents of acne patients (refer to "Chinese Clinical Dermatology" [4], meeting the diagnosis of common acne, and having more than 5 active skin lesions such as comedones, papules, pustules, cysts, nodules on the face; aged between 12 and 50 years old; not having used systemic antibiotics within 4 weeks and not having used topical antibiotics within 2 weeks), Propionibacterium acnes, Staphylococcus albus, and Staphylococcus aureus strains were cultured, isolated, and obtained respectively; in vitro antibacterial effect experiments were carried out respectively:

[0046] ① In vitro antibacterial experiment of Staphylococcus aureus: The enzyme-labeled turbidimetry was used to detect the antibacterial effect of the above experimental groups 1-5 on Staphylococcus aureus. 3 test tubes (in parallel) were taken for each group, and the treatment conditions and antibacterial results of each group of test tubes are shown in Table 3.

[0047] Table 3 In vitro antibacterial experiment of Staphylococcus aureus

[0048]

[0049] ② In vitro antibacterial experiment of Staphylococcus albus: The disk diffusion test was used to detect the antibacterial effect of the above experimental groups 1-5 on Staphylococcus albus.

[0050] Configure a 1.5×10 11 CFU / L Staphylococcus albus suspension. Use a sterile cotton swab to fully dip the bacterial suspension and coat it on MH agar. Wait for the bacterial solution to be fully absorbed. Use sterile forceps to pick up a blank drug sensitivity paper and gently place it on the agar surface, and then drop 100 μl of the composition in experimental groups 1-5 on the blank paper respectively; place the plate in a wet box, incubate at a constant temperature of 37°C for 16 hours, and then use a vernier caliper to measure the diameter of the inhibition zone, and the results are referred to Table 4.

[0051] Table 4 Results of in vitro antibacterial experiment of Staphylococcus albus

[0052] Grouping Inhibition zone diameter (mm) Experimental group 1 25.35 Experimental group 2 27.10 Experimental group 3 29.50 Experimental group 4 10.55 Experimental group 5 11.67 Control group /

[0053] ③In vitro antibacterial experiment of Propionibacterium acnes:

[0054] Prepare a suspension of Propionibacterium acnes at 1.5×10 11 CFU / L. After preparing the solid medium by the conventional method, gently place the Oxford cup on the surface of the medium with sterile forceps. Then, take 100 μl of the composition solutions of experimental groups 1-5 and add them into the Oxford cups respectively. Use a vernier caliper to accurately measure the diameter (mm) of the effective antibacterial zone of the test solution in each petri dish.

[0055] Table 5 Results of in vitro antibacterial experiment of Propionibacterium acnes

[0056] Grouping Inhibition zone diameter (mm) Experimental group 1 19.35 Experimental group 2 20.10 Experimental group 3 21.50 Experimental group 4 8.85 Experimental group 5 10.48 Control group /

[0057] It can be known from the above in vitro antibacterial experiment that both human mesenchymal stem cells alone and the hydrogel preparation alone have a certain inhibitory effect on Propionibacterium acnes, Staphylococcus albus, and Staphylococcus aureus. However, the antibacterial effect evaluation is not high. The skin care product composition prepared by combining the two has a significant inhibitory effect on the above three types of Propionibacterium acnes, Staphylococcus albus, and Staphylococcus aureus extracted and cultured from acne patients, and the antibacterial evaluation is highly effective, indicating that the extract of human mesenchymal stem cells and the active ingredients in the hydrogel have a synergistic effect.

[0058] Example 5: Animal experiment

[0059] Establish a mouse AD model by repeatedly stimulating with DNCB hapten; there are 35 mice in total, divided into experimental groups 1-5, control group 6, and sham operation group 7, with 5 mice in each group.

[0060] Modeling process: On the first day of the experiment, apply 5% DNCB (50 μl) to the shaved skin (3×6 cm 2 ) on the back of the mouse for primary sensitization, and sensitize continuously for 3 days. Then, do not do any treatment for the next 2 days. On the 6th day, change to apply 1% DNCB (50 μl) to the shaved skin on the back of the mouse for challenge, and challenge continuously for 4 days.

[0061] Treatment time and method: After modeling, apply the corresponding compositions (prepared according to the formula in Table 2 in Example 4) and solutions of experimental groups 1-5, control group 6, and sham operation group 7 to the damaged area on the back of the mouse every day. On the 4th and 10th days after modeling, detect the content levels of inflammatory factors (IL-17, TNF-α) in the serum of each group of mice (detected by ELISA kit), and the detection results are referred to Table 6, Table 7, Figure 1 and Figure 2 .

[0062] Table 6: Detection results of inflammatory factors in D4 serum

[0063] Detection of inflammatory factors in D4 serum IL-17 TNF-α Experimental group Average value N Average value N Experimental group 1 33.43 5 3.85 5 Experimental group 2 33.05 5 3.8 5 Experimental group 3 32.57 5 3.7 5 Experimental group 4 42.98 5 5.4 5 Experimental group 5 35.54 5 4.74 5 Control group 6 44.56 5 5.87 5 Sham operation group 7 21.76 5 2.49 5

[0064] Table 7: Detection results of inflammatory factors in D10 serum

[0065] Detection of inflammatory factors in D10 serum IL-17 TNF-α Experimental group Average value N Average value N Experimental group 1 21.38 5 3.15 5 Experimental group 2 21.09 5 3.1 5 Experimental group 3 20.59 5 3.02 5 Experimental group 4 30.52 5 4.39 5 Experimental group 5 29.41 5 3.57 5 Control group 6 35.27 5 5.12 5 Sham operation group 7 18.42 5 2.13 5

[0066] The detection results showed that on the 4th day after the smear treatment, the levels of inflammatory factors IL-17 and TNF-α in experimental groups 1-3 were significantly lower than those in control group 6. On the 10th day after the smear treatment, the levels of inflammatory factors IL-17 and TNF-α in experimental groups 1-3 were close to those in the sham operation group 7. Moreover, the reduction of inflammatory factor levels in experimental groups 1-3 was better than that in experimental group 4 (hydrogel preparation) and experimental group 5 (simple human mesenchymal stem cell extract). The combination of the two had an obvious synergistic promoting effect on restoring the damaged skin barrier of mice.

[0067] Although the embodiments of the present invention have been shown and described, for those of ordinary skill in the art, it can be understood that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents. Generally speaking, if those of ordinary skill in the art are inspired by it and design similar structural methods and embodiments without creative efforts without departing from the purpose of the present invention, they should all fall within the protection scope of the present invention.

Claims

1. A human umbilical cord mesenchymal stem cell extract, characterized in that Prepared by the following steps: Step 1: Resuscitate human umbilical cord mesenchymal stem cells selected from the cell bank, add them to 8 ml of DMEM culture medium containing 10% fetal bovine serum, then centrifuge at 1000 r / min, with a radius of 10 cm, for 5 min, and discard the supernatant; add 2 ml of culture medium and blow evenly, transfer the cells to a culture dish, add about 5 ml of culture medium, and culture in an incubator; Step 2: After collecting the cells, wash them once with PBS and cell lysis buffer, remove the supernatant and resuspend them in 1 volume of cell lysis buffer (generally speaking, 1uL of extract is from 10 5 Cells were obtained and lysed on ice for 30-45 min; Step 3: Use ultrasonic disruptor to disrupt cells until the cell nucleus is completely broken, centrifuge at 4℃, 15000xg for 15min, collect the supernatant, and store it in aliquots at -80℃.

2. The human umbilical cord mesenchymal stem cell extract according to claim 1, characterized in that: The cells collected in step 2 are human umbilical cord mesenchymal stem cells of passage 3-5.

3. Use of a human umbilical cord mesenchymal stem cell extract according to any one of claims 1 to 2 in the preparation of a skin care composition for soothing and repairing the skin.

4. The use according to claim 3, characterized in that: The soothing and repairing skin care composition comprises the human umbilical cord mesenchymal stem cell extract according to claim 1 and a pharmaceutically acceptable carrier, wherein the carrier is a hydrogel preparation, and the volume ratio of the human umbilical cord mesenchymal stem cell extract to the hydrogel preparation is 1:

1.

5. The use according to claim 4, characterized in that: The pH of the hydrogel preparation is 5.5-7, and each 100 ml of the hydrogel preparation includes the following ingredients: Tween 80 5-15g; Hyaluronic acid 0.5-2ml; Tea tree essence 2-8ml; Vitamin E 0.5-2ml.

6. The use according to claim 4, characterized in that: The soothing and repairing skin care product is used for soothing skin inflammation.

7. The use according to claim 4, characterized in that: The soothing and repairing skin care product is used to repair the sensitive skin barrier.

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