A composite human umbilical cord mesenchymal stem cell exosome and its application in preparing stretch mark repair suspension

Abstract

The invention discloses a composite human umbilical cord mesenchymal stem cell exosome and an application thereof in preparing a stretch mark repair suspension, belonging to the technical field of biomedicine. The invention cultured umbilical cord tissue through a first culture medium and a second culture medium to obtain the human umbilical cord mesenchymal stem cell exosome, and then mixed the human umbilical cord mesenchymal stem cell exosome with a purslane extract, squalane, tocopherol acetate and glycerol to obtain the composite human umbilical cord mesenchymal stem cell exosome, and obtained the composite human umbilical cord mesenchymal stem cell exosome with excellent stretch mark repairing effect. The human umbilical cord mesenchymal stem cell exosome can effectively improve the elasticity of the stretch mark site, reduce the stretch mark area, promote the fading of the stretch mark and reduce scars.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a composite human umbilical cord mesenchymal stem cell exosome and an application thereof in preparing a stretch mark repair suspension. Background Art

[0002] As the forefront of life science, stem cells are the focus and hotspot of global research. In recent years, with the development of medicine, many traditional stem cell therapy developers have begun to turn to the study of stem cell exosomes. Stem cell exosomes are widely used in various clinical fields because of their functions of enhancing cell vitality, inhibiting cell apoptosis, anti-inflammation, anti-fibrosis (inhibiting scar formation), antioxidant effects, promoting angiogenesis and enhancing matrix integrity.

[0003] Stretch marks, also known as skin striae or scars, are a common skin problem that forms when the skin stretches and contracts rapidly. They usually appear during pregnancy, and stretch marks are a common skin problem, especially during pregnancy, usually appearing on the abdomen, chest, buttocks, and thighs.

[0004] CN110123838B discloses the use of human pluripotent stem cell exosomes loaded with resveratrol in the preparation of drugs or cosmetics for repairing or treating stretch marks. It uses exosomes derived from embryonic stem cells and induced human pluripotent stem cells as drug carriers for resveratrol, which can greatly improve the biological effects of resveratrol. Combined with the functions of human pluripotent stem cell exosomes themselves, it can achieve better disease treatment effects. However, its preparation process is complicated and it is difficult to achieve industrial application.

[0005] CN110652491B discloses a skin care product with the effect of removing stretch marks, and its preparation method and application. Although it has a good stretch mark repairing effect, its ingredients are complex and a large amount of preservatives and niacinamide are added, which may be irritating.

[0006] In view of this, this application is filed. Summary of the invention

[0007] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a composite human umbilical cord mesenchymal stem cell exosomes and its application in the preparation of a stretch mark repair suspension. The composite human umbilical cord mesenchymal stem cell exosomes have excellent stretch mark repair effects and have broad application prospects.

[0008] To achieve the above object, the technical solution adopted by the present invention is:

[0009] A method for preparing composite human umbilical cord mesenchymal stem cell exosomes, comprising the following steps:

[0010] (1) Inoculate the processed umbilical cord tissue into the first culture medium for culture. When the cell confluence reaches 80-85%, add trypsin for digestion, and then use the first culture medium for subculture;

[0011] (2) When the third-generation cell confluence reaches 78-82%, they are transferred to the second culture medium for culture. When the cell confluence reaches 80-85%, the supernatant is collected and centrifuged to obtain human umbilical cord mesenchymal stem cell exosomes;

[0012] (3) Evenly mixing human umbilical cord mesenchymal stem cell exosomes, purslane extract, squalane, tocopherol acetate, and glycerol to obtain composite human umbilical cord mesenchymal stem cell exosomes;

[0013] The first culture medium comprises the following components: 9-11% fetal bovine serum, 10-40 μM beta-mercaptoethanol, 80-100 μg / mL glutathione, 0.01-0.04 mM aspartic acid, 10-40 ng / mL epidermal growth factor, 0.2-0.5 μg / L sodium selenite, 1-2 g / L recombinant human albumin, 0.2-0.4 mg / L anisomycin, 50-60 mg / L L-carnitine, 0.1-0.5 mg / L D-galactose, 100-200 μg / L linolenic acid, and the remainder DMEM culture medium.

[0014] The present invention creatively cultures the umbilical cord tissue with a first culture medium and a second culture medium to obtain the human umbilical cord mesenchymal stem cell exosomes, and then mixes the human umbilical cord mesenchymal stem cell exosomes with purslane extract, squalane, tocopherol acetate, and glycerol to obtain the composite human umbilical cord mesenchymal stem cell exosomes, thereby obtaining the composite human umbilical cord mesenchymal stem cell exosomes with excellent effect of repairing stretch marks. The human umbilical cord mesenchymal stem cell exosomes can effectively improve the elasticity of the stretch mark site, reduce the area of ​​the stretch mark, promote the fading of the stretch mark, and reduce scars.

[0015] The present invention effectively improves the biological activity of exosomes and the repair effect on stretch marks by optimizing the culture conditions of exosomes. The culture method of the present invention can promote cell proliferation and differentiation, protect exosomes from environmental damage during the culture process, improve their stability, and improve the purity of exosomes. The human umbilical cord mesenchymal stem cell exosomes contain rich growth factor signal molecules and complex active ingredients, which can regulate the proliferation of skin and the synthesis of collagen, effectively improve the elasticity of the skin, effectively improve the elasticity of the stretch mark area, promote skin metabolism, improve the regeneration and repair ability of skin cells, reduce collagen fiber breakage and scar formation, and accelerate the repair of stretch marks. Portulaca oleracea extract has anti-inflammatory, antioxidant and skin regeneration effects, and can accelerate the repair of stretch marks. The addition of Portulaca oleracea extract helps to improve the antioxidant stress performance of the system, improve the stability of human umbilical cord mesenchymal stem cell exosomes, and inhibit the apoptosis of keratinocytes and fibroblasts. Portulaca oleracea extract can effectively protect the skin barrier, reduce water loss, and increase the hydration rate of the skin, providing a good environment for the repair of human umbilical cord mesenchymal stem cell exosomes. At the same time, Portulaca oleracea extract has excellent soothing properties and can soothe stretch marks. The human umbilical cord mesenchymal stem cell exosomes and Portulaca oleracea extract work together, and under the synergistic effect of the two, they effectively provide a repair effect on stretch marks.

[0016] It should be noted that in the method for preparing exosomes of human umbilical cord mesenchymal stem cells, those skilled in the art can perform fluid replacement (replacement of culture medium) and oscillation (promote uniform distribution of umbilical cord tissue) according to actual conditions.

[0017] As a preferred embodiment of the present invention, the second culture medium comprises the following components: 9-11% fetal bovine serum, 10-40 μM β-mercaptoethanol, 80-100 μg / mL glutathione, 0.01-0.04 mM aspartic acid, 10-40 ng / mL epidermal growth factor, and the remainder DMEM culture medium.

[0018] As a preferred embodiment of the present invention, the culture is carried out in an incubator at 36.5-37.5°C and 5% CO2.

[0019] As a preferred embodiment of the present invention, the mass ratio of the human umbilical cord mesenchymal stem cell exosomes, purslane extract, squalane, tocopherol acetate and glycerol is 1: (0.5~2): (2~5): (2~5): (30~50).

[0020] As a preferred embodiment of the present invention, the method for preparing the purslane extract comprises the following steps:

[0021] (1) subjecting purslane powder to supercritical extraction to obtain an extraction product;

[0022] (2) adding the extraction product, cellulase, pectinase, and β-glucanase into water for enzymolysis to obtain an enzymolysis solution;

[0023] (3) first ultrafiltration with an ultrafiltration membrane with a molecular weight cutoff of 500 Da, then ultrafiltration with an ultrafiltration membrane with a molecular weight cutoff of 5000 Da, and drying to obtain a purslane extract.

[0024] The inventors of the present invention firstly used a commercially available purslane extract and found during use that the commercially available purslane extract had a certain effect in repairing stretch marks, but the effect still needed to be improved, which may be related to the extraction method of purslane.

[0025] The present invention first performs supercritical extraction on purslane, then performs enzymatic hydrolysis, and finally performs two ultrafiltrations, and the effective components with a molecular weight cutoff of 500-5000Da are more conducive to repairing stretch marks. The prepared purslane extract can well exert a synergistic effect with human umbilical cord mesenchymal stem cell exosomes, thereby improving the stretch mark repair effect.

[0026] As a preferred embodiment of the present invention, the particle size of the purslane powder is 50-400 mesh.

[0027] As a preferred embodiment of the present invention, the supercritical extraction uses CO2 as the extractant, 80~90wt% ethanol as the entrainer, the CO2 flow rate is 70~90mL / min, the extraction temperature is 38~42°C, the extraction pressure is 22~25Mpa, and the extraction time is 55~65min.

[0028] As a preferred embodiment of the present invention, the mass ratio of the extract, cellulase, pectinase, β-glucanase and water is 1: (0.01-0.03): (0.01-0.02): (0.01-0.02): (5-12).

[0029] As a preferred embodiment of the present invention, the enzymolysis temperature is 45-55°C, and the enzymolysis time is 3-7h.

[0030] The present invention also provides a medicine for repairing stretch marks, comprising the above-mentioned composite human umbilical cord mesenchymal stem cell exosomes; the medicine is a stretch mark repair suspension.

[0031] The composite human umbilical cord mesenchymal stem cell exosomes of the present invention can be added with auxiliary materials and additives to prepare a stretch mark repair suspension.

[0032] The beneficial effect of the present invention is that: the present invention cultures the umbilical cord tissue through a first culture medium and a second culture medium to obtain the human umbilical cord mesenchymal stem cell exosomes, and then mixes the human umbilical cord mesenchymal stem cell exosomes with purslane extract, squalane, tocopherol acetate, and glycerol to obtain the composite human umbilical cord mesenchymal stem cell exosomes, and obtains the composite human umbilical cord mesenchymal stem cell exosomes with excellent stretch mark repairing effect, and the human umbilical cord mesenchymal stem cell exosomes can effectively improve the elasticity of the stretch mark site, reduce the area of ​​the stretch mark, promote the fading of the stretch mark, and reduce scars. DETAILED DESCRIPTION

[0033] In order to make the purpose, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application are described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present application.

[0034] In the present application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0035] In this application, when it comes to numerical ranges, unless otherwise specified, the above numerical ranges are deemed to be continuous and include the minimum and maximum values ​​of the range, as well as each value between such minimum and maximum values. Further, when a range refers to an integer, each integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges included therein.

[0036] In the present application, there is no particular limitation on the specific dispersion and stirring treatment methods.

[0037] Unless otherwise specified, the components, raw materials or instruments used in the embodiments and comparative examples of the present invention are all commercially available raw materials or instruments, and the components and raw materials used in each parallel experiment are all of the same kind.

[0038] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In the description of the present invention, the meaning of "plurality" is at least two, such as two, three, etc., unless otherwise clearly and specifically defined.

[0039] The following examples are provided to facilitate understanding of the present invention. These examples are not provided to limit the scope of the claims.

[0040] Example 1

[0041] A method for preparing composite human umbilical cord mesenchymal stem cell exosomes, comprising the following steps:

[0042] (1) Collect umbilical cords from full-term pregnant women who underwent cesarean section and were healthy and normal, and exclude complications such as hepatitis B, hepatitis C, syphilis, AIDS, and premature rupture of membranes;

[0043] Wash with PBS buffer to remove blood from the surface of the umbilical cord and remove the artery and vein;

[0044] The umbilical cord was cut into several small pieces and washed, and then Wharton's jelly was torn off and fully cut into small pieces of 1±0.05 cm to obtain the processed umbilical cord tissue;

[0045] (2) Add 2±0.05 g of processed umbilical cord tissue and 20 mL of the first culture medium to a culture bottle, place in an incubator, and culture at 37°C and 5% CO2 (during the culture process, the conditions remain unchanged, the same below), and replace 10 mL of the first culture medium every 3 days; when the cell confluence reaches 85%, add 0.2 wt% trypsin for digestion, replace the first culture medium in the culture bottle, and use the first culture medium for subculture;

[0046] During the culture process, the umbilical cord tissue blocks can be evenly distributed by shaking as needed;

[0047] Each 1L of the first culture medium includes the following components: 10% fetal bovine serum, 20μM β-mercaptoethanol, 90μg / mL glutathione, 0.02mM aspartic acid, 20ng / mL epidermal growth factor, 0.4μg / L sodium selenite, 1.6g / L recombinant human albumin, 0.3mg / L anisomycin, 55mg / L L-carnitine, 0.2mg / L D-galactose, 150μg / L linolenic acid, and the remainder DMEM culture medium.

[0048] (3) When the cell confluence of the third generation cells reached 80%, they were transferred to the second culture medium for culture. When the cell confluence reached 85%, the supernatant was collected and filtered with 0.45 μm and 0.22 μm filters respectively. The filtered supernatant was collected and ultracentrifuged at 80,000 g for 30 min. The precipitate was collected to obtain human umbilical cord mesenchymal stem cell exosomes.

[0049] The second culture medium includes the following components: 10% fetal bovine serum, 30 μM β-mercaptoethanol, 90 μg / mL glutathione, 0.03 mM aspartic acid, 25 ng / mL epidermal growth factor, and the remainder DMEM culture medium.

[0050] (4) Mix human umbilical cord mesenchymal stem cell exosomes, purslane extract, squalane, tocopherol acetate, and glycerol in a mass ratio of 1:0.5:5:2:41.5 to obtain composite human umbilical cord mesenchymal stem cell exosomes.

[0051] Wherein, the preparation method of the purslane extract comprises the following steps:

[0052] (1) Grinding purslane into 200 mesh to obtain purslane powder; adding the purslane powder into an extraction axe to perform supercritical extraction to obtain an extraction product; the supercritical extraction uses CO2 as an extractant, 85wt% ethanol as an entrainer, a CO2 flow rate of 80mL / min, an extraction temperature of 40°C, an extraction pressure of 24Mpa, and an extraction time of 60min.

[0053] (2) The extract, cellulase, pectinase and β-glucanase are added to water, and enzymolysis is carried out at 50° C. for 5 hours to obtain an enzymolysis solution; the mass ratio of the extract, cellulase, pectinase, β-glucanase and water is 1:0.03:0.02:0.01:10.

[0054] (3) first ultrafiltration with an ultrafiltration membrane with a molecular weight cutoff of 500 Da, then ultrafiltration with an ultrafiltration membrane with a molecular weight cutoff of 5000 Da, and drying to obtain a purslane extract.

[0055] Example 2

[0056] A method for preparing composite human umbilical cord mesenchymal stem cell exosomes, comprising the following steps:

[0057] (1) Collect umbilical cords from full-term pregnant women who underwent cesarean section and were healthy and normal, and exclude complications such as hepatitis B, hepatitis C, syphilis, AIDS, and premature rupture of membranes;

[0058] Wash with PBS buffer to remove blood from the surface of the umbilical cord and remove the artery and vein;

[0059] The umbilical cord was cut into several small pieces and washed, and then Wharton's jelly was torn off and fully cut into small pieces of 1±0.05 cm to obtain the processed umbilical cord tissue;

[0060] (2) Add 2±0.05 g of processed umbilical cord tissue and 20 mL of the first culture medium to a culture bottle, place in an incubator, and culture at 37°C and 5% CO2 (during the culture process, the conditions remain unchanged, the same below), and replace 10 mL of the first culture medium every 3 days; when the cell confluence reaches 85%, add 0.2 wt% trypsin for digestion, replace the first culture medium in the culture bottle, and use the first culture medium for subculture;

[0061] During the culture process, the umbilical cord tissue blocks can be evenly distributed by shaking as needed;

[0062] Each 1L of the first culture medium includes the following components: 10% fetal bovine serum, 20μM β-mercaptoethanol, 90μg / mL glutathione, 0.02mM aspartic acid, 20ng / mL epidermal growth factor, 0.4μg / L sodium selenite, 1.6g / L recombinant human albumin, 0.3mg / L anisomycin, 55mg / L L-carnitine, 0.2mg / L D-galactose, 150μg / L linolenic acid, and the remainder DMEM culture medium.

[0063] (3) When the cell confluence of the third generation cells reached 80%, they were transferred to the second culture medium for culture. When the cell confluence reached 85%, the supernatant was collected and filtered with 0.45 μm and 0.22 μm filters respectively. The filtered supernatant was collected and ultracentrifuged at 80,000 g for 30 min. The precipitate was collected to obtain human umbilical cord mesenchymal stem cell exosomes.

[0064] The second culture medium includes the following components: 10% fetal bovine serum, 30 μM β-mercaptoethanol, 90 μg / mL glutathione, 0.03 mM aspartic acid, 25 ng / mL epidermal growth factor, and the remainder DMEM culture medium.

[0065] (4) Mix human umbilical cord mesenchymal stem cell exosomes, purslane extract, squalane, tocopherol acetate, and glycerol in a mass ratio of 1:2:2:5:40 to obtain composite human umbilical cord mesenchymal stem cell exosomes.

[0066] Wherein, the preparation method of the purslane extract comprises the following steps:

[0067] (1) Grinding purslane into 200 mesh to obtain purslane powder; adding the purslane powder into an extraction axe to perform supercritical extraction to obtain an extraction product; the supercritical extraction uses CO2 as an extractant, 85wt% ethanol as an entrainer, a CO2 flow rate of 80mL / min, an extraction temperature of 40°C, an extraction pressure of 24Mpa, and an extraction time of 60min.

[0068] (2) The extract, cellulase, pectinase and β-glucanase are added to water, and enzymolysis is carried out at 50° C. for 5 hours to obtain an enzymolysis solution; the mass ratio of the extract, cellulase, pectinase, β-glucanase and water is 1:0.03:0.02:0.01:10.

[0069] (3) first ultrafiltration with an ultrafiltration membrane with a molecular weight cutoff of 500 Da, then ultrafiltration with an ultrafiltration membrane with a molecular weight cutoff of 5000 Da, and drying to obtain a purslane extract.

[0070] Example 3

[0071] A method for preparing composite human umbilical cord mesenchymal stem cell exosomes, comprising the following steps:

[0072] (1) Collect umbilical cords from full-term pregnant women who underwent cesarean section and were healthy and normal, and exclude complications such as hepatitis B, hepatitis C, syphilis, AIDS, and premature rupture of membranes;

[0073] Wash with PBS buffer to remove blood from the surface of the umbilical cord and remove the artery and vein;

[0074] The umbilical cord was cut into several small pieces and washed, and then Wharton's jelly was torn off and fully cut into small pieces of 1±0.05 cm to obtain the processed umbilical cord tissue;

[0075] (2) Add 2±0.05 g of processed umbilical cord tissue and 20 mL of the first culture medium to a culture bottle, place in an incubator, and culture at 37°C and 5% CO2 (during the culture process, the conditions remain unchanged, the same below), and replace 10 mL of the first culture medium every 3 days; when the cell confluence reaches 85%, add 0.2 wt% trypsin for digestion, replace the first culture medium in the culture bottle, and use the first culture medium for subculture;

[0076] During the culture process, the umbilical cord tissue blocks can be evenly distributed by shaking as needed;

[0077] Each 1L of the first culture medium includes the following components: 10% fetal bovine serum, 20μM β-mercaptoethanol, 90μg / mL glutathione, 0.02mM aspartic acid, 20ng / mL epidermal growth factor, 0.4μg / L sodium selenite, 1.6g / L recombinant human albumin, 0.3mg / L anisomycin, 55mg / L L-carnitine, 0.2mg / L D-galactose, 150μg / L linolenic acid, and the remainder DMEM culture medium.

[0078] (3) When the cell confluence of the third generation cells reached 80%, they were transferred to the second culture medium for culture. When the cell confluence reached 85%, the supernatant was collected and filtered with 0.45 μm and 0.22 μm filters respectively. The filtered supernatant was collected and ultracentrifuged at 80,000 g for 30 min. The precipitate was collected to obtain human umbilical cord mesenchymal stem cell exosomes.

[0079] The second culture medium includes the following components: 10% fetal bovine serum, 30 μM β-mercaptoethanol, 90 μg / mL glutathione, 0.03 mM aspartic acid, 25 ng / mL epidermal growth factor, and the remainder DMEM culture medium.

[0080] (4) Mix human umbilical cord mesenchymal stem cell exosomes, purslane extract, squalane, tocopherol acetate, and glycerol in a mass ratio of 1:0.5:5:2:41.5 to obtain composite human umbilical cord mesenchymal stem cell exosomes.

[0081] Wherein, the preparation method of the purslane extract comprises the following steps:

[0082] (1) Grinding purslane into 200 mesh to obtain purslane powder; adding the purslane powder into an extraction axe to perform supercritical extraction to obtain an extraction product; the supercritical extraction uses CO2 as an extractant, 85wt% ethanol as an entrainer, a CO2 flow rate of 80mL / min, an extraction temperature of 40°C, an extraction pressure of 24Mpa, and an extraction time of 60min.

[0083] (2) The extract, cellulase, pectinase and β-glucanase are added to water, and enzymolysis is carried out at 50° C. for 5 hours to obtain an enzymolysis solution; the mass ratio of the extract, cellulase, pectinase, β-glucanase and water is 1:0.01:0.01:0.02:10.

[0084] (3) first ultrafiltration with an ultrafiltration membrane with a molecular weight cutoff of 500 Da, then ultrafiltration with an ultrafiltration membrane with a molecular weight cutoff of 5000 Da, and drying to obtain a purslane extract.

[0085] Comparative Example 1

[0086] The difference between Comparative Example 1 and Example 1 is that Comparative Example 1 does not contain the purslane extract (replaced with an equal amount of human umbilical cord mesenchymal stem cell exosomes), and the rest is the same.

[0087] Human umbilical cord mesenchymal stem cell exosomes, squalane, tocopherol acetate and glycerol were evenly mixed in a mass ratio of 1.5:5:2:41.5 to obtain composite human umbilical cord mesenchymal stem cell exosomes.

[0088] Comparative Example 2

[0089] The difference between Comparative Example 2 and Example 1 is that Comparative Example 2 does not contain human umbilical cord mesenchymal stem cell exosomes (replaced by an equal amount of Portulaca oleracea extract), and the rest are the same.

[0090] The purslane extract, squalane, tocopherol acetate and glycerin were uniformly mixed in a mass ratio of 1.5:5:2:41.5 to obtain a mixed solution.

[0091] Comparative Example 3

[0092] The difference between Comparative Example 3 and Example 1 is that Comparative Example 3 uses commercially available purslane extract to replace the purslane extract of the present invention.

[0093] The purslane extract of this comparative example was purchased from Ruiyu Chemical with the brand name of HerbEx Protulaca.

[0094] Comparative Example 4

[0095] The difference between Comparative Example 4 and Example 1 is that Comparative Example 4 uses an equal amount of Centella asiatica extract to replace the Portulaca oleracea extract, and the other contents are the same.

[0096] Human umbilical cord mesenchymal stem cell exosomes, Centella asiatica extract, squalane, tocopherol acetate and glycerol were evenly mixed in a mass ratio of 1:0.5:5:2:41.5 to obtain composite human umbilical cord mesenchymal stem cell exosomes.

[0097] The Centella asiatica extract was purchased from Chennuo Biotech, brand name Hoshea CNCA.

[0098] Comparative Example 5

[0099] The difference between Comparative Example 5 and Example 1 is that Comparative Example 5 uses an equal amount of Centella asiatica extract to replace human umbilical cord mesenchymal stem cell exosomes, and the rest are the same.

[0100] Centella asiatica extract, Portulaca oleracea extract, squalane, tocopherol acetate and glycerol were uniformly mixed in a mass ratio of 1:0.5:5:2:41.5 to obtain composite human umbilical cord mesenchymal stem cell exosomes.

[0101] The Centella asiatica extract was purchased from Chennuo Biotech, brand name Hoshea CNCA.

[0102] Comparative Example 6

[0103] The difference between Comparative Example 6 and Example 1 is that the mass ratio of human umbilical cord mesenchymal stem cell exosomes and Portulaca oleracea extract is not within the scope of the present invention, and the others are the same.

[0104] Human umbilical cord mesenchymal stem cell exosomes, purslane extract, squalane, tocopherol acetate and glycerol were evenly mixed in a mass ratio of 1.3:0.2:5:2:41.5 to obtain composite human umbilical cord mesenchymal stem cell exosomes.

[0105] Comparative Example 7

[0106] The difference between Comparative Example 7 and Example 1 is that the mass ratio of human umbilical cord mesenchymal stem cell exosomes and Portulaca oleracea extract is not within the scope of the present invention, and the others are the same.

[0107] Human umbilical cord mesenchymal stem cell exosomes, purslane extract, squalane, tocopherol acetate and glycerol were evenly mixed in a mass ratio of 0.2:1.3:5:2:41.5 to obtain composite human umbilical cord mesenchymal stem cell exosomes.

[0108] Test Case

[0109] 1. Security Testing

[0110] 1.1 The experimental animals were New Zealand white rabbits of normal grade, purchased from Guangdong Medical Experimental Animal Center.

[0111] 1.2 Select 12 New Zealand white rabbits and divide them into three groups, each with 4 groups. Remove the hair on both sides of the spine of the New Zealand rabbits with scissors, and the hair removal range is 5*5cm on each side;

[0112] 1.3 2 mL of the product prepared in Examples 1 to 3 (all applied on the left side, with the right side as a control) was evenly applied to the left and right hair removal areas of New Zealand rabbits, covered with gauze and cellophane, and fixed with a bandage. The covering was removed after 6 hours, and the test area and the control area were compared after 24 hours. No abnormalities were found, and no redness, swelling, or other abnormalities were observed, indicating that the product of the present invention had no irritation or allergic reaction and had good safety.

[0113] 2. Efficacy test

[0114] 2.1 80 6-week-old C57 / BL6J mice (male) were randomly divided into 10 groups, with 8 mice in each group.

[0115] 2.2 The mice were anesthetized, the hair on the left side of the spine was shaved, and the skin with an area of ​​1*1 cm was carefully cut off. The wound was fixed with sterile thread and a dressing was applied. After 1 day, the dressing was removed and the products of the embodiment and the comparative example were applied to the wound. The application amount was 0.2 mL / mouse, and the application was continued for two weeks.

[0116] 2.3 Calculate the wound healing rate using the formula: (1-unhealed wound area / original wound area) × 100%.

[0117] 2.4 Calculate scar area.

[0118] Table 1

[0119]

[0120] 3. Efficacy test

[0121] 3.1 100 volunteers were selected. All volunteers were 0.5 to 3 years postpartum and had obvious stretch marks on their abdomens. All volunteers were able to cooperate well with the experiment and did not accept other stretch mark products or other stretch mark treatments during the trial.

[0122] 3.2 100 volunteers were randomly divided into 10 groups, with 10 people in each group. Before the test, the skin elasticity Q1 was tested using a skin elasticity tester Cutometer (Courage & Khazaka, Germany). The products of the embodiment and the comparative example were used in the morning and evening every day. After 4 weeks, the skin elasticity Q1 was tested again.

[0123] 3.3 Calculation of skin elasticity improvement rate: The calculation formula is: (skin elasticity after use Q1 / skin elasticity before use-1)×100%.

[0124] 3.4 Rating: The total score of each usage effect is 5 points (1-5 points, each person's score is a positive integer): 5 points is the highest score, indicating good and very satisfactory; 4 points is very good; 3 points is acceptable; and below 3 points is not good and unacceptable. Calculate the total score.

[0125] Table 2

[0126]

[0127] As can be seen from Table 1, the composite human umbilical cord mesenchymal stem cell exosomes of the present invention have an excellent effect on repairing stretch marks.

[0128] By comparing Example 1 with Comparative Examples 1 to 5, it can be seen that the combination of the human umbilical cord mesenchymal stem cell exosomes of the present invention and the Portulaca oleracea extract has a significant synergistic effect. Under the joint action of the two, the repair effect of stretch marks is significantly improved.

[0129] By comparing Example 1 with Comparative Examples 6 to 7, it can be seen that the present invention further improves the repair effect of stretch marks by controlling the dosage of human umbilical cord mesenchymal stem cell exosomes and the purslane extract within the scope of the present invention.

[0130] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, 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 scope of the technical solution of the present invention.

Claims

1. A method for preparing composite human umbilical cord mesenchymal stem cell exosomes, characterized in that: The following steps are involved: (1) Inoculate the processed umbilical cord tissue into the first culture medium for culture. When the cell confluence reaches 80-85%, add trypsin for digestion, and then use the first culture medium for subculture; (2) When the third-generation cell confluence reaches 78-82%, transfer to the second culture medium for culture. When the cell confluence reaches 80-85%, collect the supernatant, filter, and centrifuge to obtain human umbilical cord mesenchymal stem cell exosomes; (3) Mixing human umbilical cord mesenchymal stem cell exosomes, purslane extract, squalane, tocopherol acetate, and glycerol in a mass ratio of 1: (0.5-2): (2-5): (2-5): (30-50) to obtain composite human umbilical cord mesenchymal stem cell exosomes; The first culture medium comprises the following components: 9-11% fetal bovine serum, 10-40 μM β-mercaptoethanol, 80-100 μg / mL glutathione, 0.01-0.04 mM aspartic acid, 10-40 ng / mL epidermal growth factor, 0.2-0.5 μg / L sodium selenite, 1-2 g / L recombinant human albumin, 0.2-0.4 mg / L anisomycin, 50-60 mg / L L-carnitine, 0.1-0.5 mg / L D-galactose, 100-200 μg / L linolenic acid, and the remainder DMEM culture medium; The preparation method of the purslane extract comprises the following steps: (1) subjecting purslane powder to supercritical extraction to obtain an extraction product; (2) adding the extraction product, cellulase, pectinase, and β-glucanase to water for enzymatic hydrolysis to obtain an enzymatic hydrolyzate; and (3) firstly ultrafiltering with an ultrafiltration membrane having a molecular weight cutoff of 500Da, and then ultrafiltering with an ultrafiltration membrane having a molecular weight cutoff of 5000Da, and drying to obtain the purslane extract.

2. The method for preparing composite human umbilical cord mesenchymal stem cell exosomes according to claim 1, characterized in that: The second culture medium comprises the following components: 9-11% fetal bovine serum, 10-40 μM beta-mercaptoethanol, 80-100 μg / mL glutathione, 0.01-0.04 mM aspartic acid, 10-40 ng / mL epidermal growth factor, and the remainder DMEM culture medium.

3. The method for preparing composite human umbilical cord mesenchymal stem cell exosomes according to claim 1, characterized in that: The culture was carried out in an incubator at 36.5-37.5°C and 5% CO2.

4. The method for preparing composite human umbilical cord mesenchymal stem cell exosomes according to claim 1, characterized in that: The particle size of the purslane powder is 50-400 meshes.

5. The method for preparing composite human umbilical cord mesenchymal stem cell exosomes according to claim 1, characterized in that: The supercritical extraction uses CO2 as the extractant, 80-90wt% ethanol as the entrainer, the CO2 flow rate is 70-90mL / min, the extraction temperature is 38-42°C, the extraction pressure is 22-25Mpa, and the extraction time is 55-65min.

6. The method for preparing composite human umbilical cord mesenchymal stem cell exosomes according to claim 1, characterized in that: The mass ratio of the extraction product, cellulase, pectinase, β-glucanase and water is 1: (0.01-0.03): (0.01-0.02): (0.01-0.02): (5-12).

7. The method for preparing composite human umbilical cord mesenchymal stem cell exosomes according to claim 1, characterized in that: The enzymolysis temperature is 45-55° C., and the enzymolysis time is 3-7 hours.

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