Composition for promoting cell regeneration and application thereof

Abstract

The present invention relates to a composition for promoting cell regeneration and its application. Specifically, a composition for promoting cell regeneration is provided, comprising L-tryptophanol oxalate, isosorbide-2-acetate, and glabridin. The composition is used to prepare a cell regeneration agent and construct a cell model. The composition can effectively enhance cell proliferation and CD63 expression levels in cells. The present invention not only effectively enhances cell regeneration capacity, but also is simple to operate and low in cost. It can be applied to the preparation of cosmetics, skin care products, wound repair materials or drugs, and skin repair.

Description

Technical Field

[0001] The present invention relates to a composition having a beneficial effect on the skin, and in particular to a composition for promoting cell regeneration and an application thereof. Background Art

[0002] Currently, for women, the gradual aging of their skin is undoubtedly an unpleasant loss of elegance. Skin aging is a phenomenon caused by both natural and unnatural factors. After birth, skin tissue develops and becomes increasingly functional, but begins to degenerate at a certain age. This is a natural phenomenon of skin aging. Unnatural factors that cause skin aging include ultraviolet radiation, pollution, emotions, and stress. In addition, there are many other causes of skin damage, such as burns, genetic diseases, and chronic trauma. Foot ulcers are common in diabetics. Due to the diabetic microenvironment, these skin ulcers are difficult to heal and there is no effective treatment. Therefore, many skin ulcers in the human body cannot recover on their own after being damaged, and large-scale skin damage is highly susceptible to infection and death. Therefore, the invention of a composition with beneficial effects on the skin is of great significance in the field of skin repair. Summary of the Invention

[0003] The purpose of the present invention is to provide a composition for promoting cell regeneration and its application.

[0004] The technical solutions adopted by the present invention to achieve the above-mentioned purpose are:

[0005] A composition comprises at least one of L-tryptophanol oxalate and isosorbide-2-acetate. Among them, when the composition is L-tryptophanol oxalate and isosorbide-2-acetate, when added to F12 complete medium as a cell regeneration agent for cell culture, the use of L-tryptophanol oxalate and isosorbide-2-acetate significantly increased the expression level of CD63 in the cells, indicating that L-tryptophanol oxalate and isosorbide-2-acetate can effectively increase the secretion of exosomes, and at the same time can effectively improve wound recovery ability and promote cell regeneration ability; in addition, when the composition is a single L-tryptophanol oxalate or isosorbide-2-acetate, when added to F12 complete medium as a cell regeneration agent for cell culture, the use of a single L-tryptophanol oxalate or isosorbide-2-acetate has no significant effect on improving the expression level of CD63 in the cells, indicating that the use of a single L-tryptophanol oxalate or isosorbide-2-acetate cannot effectively increase the secretion of exosomes, and has no significant promoting effect on wound recovery ability and cell regeneration ability.

[0006] Preferably, L-tryptophanol oxalate and isosorbide-2-acetate are mixed in a mass ratio of 1:1 to 4.

[0007] Preferably, a composition comprises at least one of L-tryptophanol oxalate, isosorbide-2-acetate and glabridol.

[0008] More preferably, the composition comprises L-tryptophanol oxalate.

[0009] More preferably, the composition comprises L-tryptophanol oxalate and isosorbide-2-acetate.

[0010] More preferably, the mass ratio of L-tryptophanol oxalate to isosorbide-2-acetate is 1:1-4.

[0011] More preferably, the composition includes L-tryptophanol oxalate, isosorbide-2-acetate, and glabripenol. When glabripenol is added to F12 complete medium containing L-tryptophanol oxalate and isosorbide-2-acetate as a cell regeneration agent for cell culture, the use of glabripenol further significantly increases the expression level of CD63 in the cells, indicating that glabripenol can further effectively increase the secretion of exosomes, while effectively improving wound recovery and promoting cell regeneration.

[0012] More preferably, the mass ratio of L-tryptophanol oxalate, isosorbide-2-acetate and glabriquitin is 1:1-4:1-4.

[0013] The present invention also provides a cell regeneration agent, comprising: the above composition.

[0014] Preferably, the cell regeneration agent further comprises F12 complete medium, and the concentration of the composition in the F12 complete medium is 0.002-0.008 g / ml.

[0015] The present invention also provides a method for constructing a cell model, comprising: culturing cells using the above-mentioned cell regeneration agent to obtain a cell model.

[0016] Preferably, the cells include any one of hematopoietic stem cells, adipose stem cells, umbilical cord blood stem cells, and umbilical cord mesenchymal stem cells.

[0017] Preferably, the exosomes secreted by the cells in the cell model contain at least one of nucleic acid, protein and cholesterol.

[0018] More preferably, the surface markers of exosomes include at least one of CD63, CD81, CD9, TSG101 and HSP27.

[0019] Preferably, a method for constructing a cell model is as follows:

[0020] S1: Cells were cultured with F12 complete medium.

[0021] S2: The supernatant of the cells in the growth phase in S1 was removed, the cells were washed with PBS, and trypsin was digested. F12 complete medium was added to obtain a cell suspension, and the cell suspension was centrifuged to obtain cell 1.

[0022] S3: Subculture: Cell 1 is mixed with F12 complete medium and cultured at a constant temperature to obtain cell 2.

[0023] S4: Cell 2 is subcultured according to the proportion, a cell regeneration agent is added, and the cells are cultured at a constant temperature. At the end of the culture, the cells are washed with PBS to obtain a cell model.

[0024] More preferably, the cells are stem cells, including any one of hematopoietic stem cells, adipose stem cells, umbilical cord blood stem cells, and umbilical cord mesenchymal stem cells.

[0025] More preferably, the stem cells are umbilical cord mesenchymal stem cells.

[0026] More preferably, the culture time in S1 is 10 to 14 hours.

[0027] More preferably, the amount of PBS used for washing cells in S2 is 4 to 6 parts by volume.

[0028] More preferably, the mass percentage of pancreatic enzyme in S2 is 0.2-0.3%, the amount of pancreatic enzyme used is 0.5-1.5 parts by volume, and the digestion time is 1-3 minutes.

[0029] More preferably, the amount of F12 complete medium in S2 is 3 to 5 parts by volume.

[0030] More preferably, the centrifugal speed in S2 is 1000-2000 r / min, and the centrifugal time is 4-6 min.

[0031] More preferably, the amount of F12 complete medium in S3 is 8 to 12 parts by volume.

[0032] More preferably, the constant temperature of culture in S3 is 35-38°C.

[0033] More preferably, the ratio in S4 is 1:0.5~1.5.

[0034] More preferably, the amount of the cell regeneration agent in S4 is 0.2 to 0.8 parts by volume.

[0035] More preferably, the cell regeneration agent in S4 further comprises F12 complete culture medium, and the concentration of the composition in the F12 complete culture medium is 0.002-0.008 g / ml.

[0036] More preferably, the culture time in S4 is 15 to 25 hours.

[0037] More preferably, the constant temperature culture temperature in S4 is 35-39°C.

[0038] More preferably, the amount of PBS used for washing cells in S4 is 4 to 6 parts by volume, and the number of washes is 2 to 4 times.

[0039] The present invention also provides use of the composition in preparing a cell model and / or a wound repair material and / or a medicine and / or a wound recovery agent.

[0040] The present invention also provides use of the cell regeneration agent in preparing a cell model and / or a wound repair material and / or a medicine and / or a wound recovery agent.

[0041] The present invention utilizes a cell regeneration agent prepared by adding the composition to F12 complete culture medium and applying it to the skin repair field, thereby achieving the following beneficial effects: the composition not only effectively enhances the regenerative capacity of cells, but also is simple to operate, low-cost, and widely applicable to the skin repair field. Therefore, the present invention provides a composition for promoting cell regeneration and its application. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 The graph shows the absorbance measurement results using a microplate reader.

[0043] Figure 2 This is a graph showing the wound recovery rate in rats.

[0044] Figure 3 The graph shows the expression results of CD63 in umbilical cord mesenchymal stem cells. DETAILED DESCRIPTION

[0045] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0046] The following first describes the concepts involved in this application with reference to the accompanying drawings. It should be noted that the following description of each concept is intended only to make the content of this application easier to understand and does not limit the scope of protection of this application. At the same time, the embodiments and features in the embodiments of this application can be combined with each other unless there is a conflict. The following detailed description of this application will be made with reference to the accompanying drawings and in conjunction with the embodiments.

[0047] Example 1:

[0048] A composition for promoting cell regeneration comprises L-tryptophanol oxalate and isosorbide-2-acetate, wherein the mass ratio of L-tryptophanol oxalate to isosorbide-2-acetate is 1:1.

[0049] Example 2:

[0050] This embodiment differs from embodiment 1 in that the mass ratio of L-tryptophanol oxalate to isosorbide-2-acetate is 1:4.

[0051] Example 3:

[0052] The difference between this embodiment and embodiment 2 is that glabridol is added during the preparation of the composition, specifically:

[0053] A composition for promoting cell regeneration comprises L-tryptophanol oxalate, isosorbide-2-acetate and glabridin, wherein the mass ratio of L-tryptophanol oxalate to isosorbide-2-acetate and glabridin is 1:4:1.

[0054] Example 4:

[0055] The difference between this embodiment and embodiment 3 is that the mass ratio of L-tryptophanol oxalate, isosorbide-2-acetate, and glabriquitin is 1:4:4.

[0056] Example 5:

[0057] A cell regeneration agent comprises: the composition prepared in Example 1 and F12 complete medium. The amount of the composition is 0.05 g, and the amount of the F12 complete medium is 10 ml.

[0058] Example 6:

[0059] A cell regeneration agent. This embodiment differs from embodiment 5 in that the composition is the composition of embodiment 2.

[0060] Example 7:

[0061] A cell regeneration agent. This embodiment differs from embodiment 5 in that the composition is the composition of embodiment 3.

[0062] Example 8:

[0063] A cell regeneration agent. This embodiment differs from embodiment 5 in that the composition is the composition of embodiment 4.

[0064] Comparative Example 1:

[0065] A cell regeneration agent comprises: L-tryptophanol oxalate and F12 complete culture medium. The amount of L-tryptophanol oxalate used is 0.05 g, and the amount of F12 complete culture medium used is 10 ml.

[0066] Comparative Example 2:

[0067] A cell regeneration agent comprises: isosorbide-2-acetate and F12 complete medium. The amount of isosorbide-2-acetate used is 0.05g, and the amount of F12 complete medium used is 10ml.

[0068] The cell counting method was used to measure the proliferation rate of mouse fibroblasts (L-929 cells) produced by the cell regeneration agent prepared in Example 5, thereby conducting a safety assessment of the toxicity of the cell regeneration agent. The results showed that the cell regeneration agent significantly promoted the proliferation of mouse fibroblasts (L-929 cells). Therefore, the cell regeneration agent prepared by the present invention is practically non-toxic.

[0069] Example 9:

[0070] A method for constructing a cell model comprises the following steps:

[0071] (1) Culture umbilical cord mesenchymal stem cells in F12 complete medium for 12 hours.

[0072] (2) Remove the supernatant from the umbilical cord mesenchymal stem cells in the growth phase, aspirate PBS to wash the umbilical cord mesenchymal stem cells, add trypsin for digestion, invert and observe when the umbilical cord mesenchymal stem cells are in a free state, aspirate F12 complete medium and add it to the cell bottle, gently pipette to make an umbilical cord mesenchymal stem cell suspension, and then centrifuge. The amount of PBS used to wash the umbilical cord mesenchymal stem cells is 5 mL, the mass percentage of trypsin used to digest the umbilical cord mesenchymal stem cells is 0.25%, the amount of trypsin used is 1 mL, the digestion time is 2 min, the amount of F12 complete medium used is 4 mL, the centrifugal speed is 1500 r / min, and the centrifugation time is 5 min.

[0073] (3) Add F12 complete medium at a ratio of 1 to 2, mix thoroughly by pipetting, and then culture in a constant temperature incubator. The amount of F12 complete medium used is 10 mL, and the constant temperature is 37°C.

[0074] (4) At a ratio of 1:1, the umbilical cord mesenchymal stem cells were cultured in a cell flask for a certain period of time and subcultured into a 6-well cell culture plate. F12 complete medium was added to each cell well and cultured in a constant temperature incubator. The umbilical cord mesenchymal stem cells were washed with PBS to obtain an umbilical cord mesenchymal stem cell model. The umbilical cord mesenchymal stem cells were cultured in the cell flask for 20 hours, the amount of F12 complete medium used was 0.5 mL, the constant temperature was 37°C, the amount of PBS used to wash the umbilical cord mesenchymal stem cells was 5 mL, and the PBS was washed three times.

[0075] Example 10:

[0076] A method for constructing a cell model.

[0077] The difference between this embodiment and embodiment 9 is that the F12 complete culture medium in step (4) of embodiment 9 is replaced with the cell regeneration agent in embodiment 5. Other than that, the other conditions are the same.

[0078] Example 11:

[0079] A method for constructing a cell model.

[0080] The difference between this embodiment and embodiment 9 is that the F12 complete culture medium in step (4) of embodiment 9 is replaced with the cell regeneration agent in embodiment 6. Other than that, the other conditions are the same.

[0081] Example 12:

[0082] A method for constructing a cell model.

[0083] The difference between this embodiment and embodiment 9 is that the F12 complete culture medium in step (4) of embodiment 9 is replaced with the cell regeneration agent in embodiment 7. Other than that, the other conditions are the same.

[0084] Example 13:

[0085] A method for constructing a cell model.

[0086] The difference between this embodiment and embodiment 9 is that the F12 complete culture medium in step (4) of embodiment 9 is replaced with the cell regeneration agent in embodiment 8. Other than that, the other conditions are the same.

[0087] Comparative Example 3:

[0088] A method for constructing a cell model.

[0089] The difference between this embodiment and embodiment 9 is that the F12 complete culture medium in step (4) of embodiment 9 is replaced with the cell regeneration agent in comparative example 1. Other than that, the other conditions are the same.

[0090] Comparative Example 4:

[0091] A method for constructing a cell model.

[0092] The difference between this embodiment and embodiment 9 is that the F12 complete culture medium in step (4) of embodiment 9 is replaced with the cell regeneration agent in comparative example 2. Other than that, the other conditions are the same.

[0093] Experimental Example 1

[0094] Cells: Umbilical cord mesenchymal stem cells

[0095] Experimental group: umbilical cord mesenchymal stem cells cultured in Examples 9-13

[0096] Control group: umbilical cord mesenchymal stem cells cultured in comparative examples 3-4

[0097] Experimental instrument: microplate reader

[0098] The experimental process and specific methods are as follows:

[0099] Umbilical cord mesenchymal stem cells that have been continuously cultured to the fourth generation were selected and inoculated into the well plate according to a certain number of umbilical cord mesenchymal stem cells / well. F12 complete medium was added and the F12 complete medium was evenly divided into 3 groups, respectively recorded as A, B, and C. The umbilical cord mesenchymal stem cells in the three groups of F12 complete medium were cultured in the incubator for a certain period of time. After the culture was completed, the proliferation of the umbilical cord mesenchymal stem cells was examined using the CCK8 kit. Before the measurement, the F12 complete medium was replaced with serum-free DMEM medium, the serum-free DMEM medium and the CCK8 solution were mixed evenly, and the culture was continued in the incubator. The absorbance was measured using a microplate reader. The results are shown in the figure ( Figure 1 ). The number of umbilical cord mesenchymal stem cells was 4000. Group A was cultured in an incubator for 48 hours, Group B was cultured for 96 hours, and Group C was cultured for 144 hours. The amount of serum-free DMEM medium used was 100 μL, the amount of CCK8 solution used was 10 μL, the culture temperature was 37°C, the culture time was 2 hours, and the measurement wavelength was 450 nm.

[0100] from Figure 1 It can be seen that at each time point, the data results of the three groups of Examples 9 to 11 are compared, and the data of Example 9 is smaller, indicating that the addition of L-tryptophanol oxalate and isosorbide-2-acetate to the F12 complete medium can effectively promote the proliferation of umbilical cord mesenchymal stem cells, and further illustrate that the addition of L-tryptophanol oxalate and isosorbide-2-acetate to the F12 complete medium is beneficial to promoting cell regeneration; at the same time, the higher the amount of L-tryptophanol oxalate and isosorbide-2-acetate, the more conducive to promoting cell regeneration. The data results of the three groups of Examples 11 to 13 are compared, and the data of Examples 12 and 13 are larger, indicating that the addition of glabripenol to the F12 complete medium can effectively promote the proliferation of umbilical cord mesenchymal stem cells, and further illustrate that the addition of glabripenol to the F12 complete medium is beneficial to promoting cell regeneration; at the same time, the higher the amount of glabripenol, the more conducive to promoting cell regeneration. Comparing the data results of Example 10 and Comparative Examples 3-4, the data of Example 10 is higher, indicating that adding L-tryptophanol oxalate or isosorbide-2-acetate alone to F12 complete culture medium is not conducive to promoting cell regeneration.

[0101] Experimental Example 2

[0102] Experimental group: Cell regeneration agent of Examples 5-8

[0103] Control group: cell regeneration agent of comparative example 1-2

[0104] Blank group: natural growth

[0105] Experimental instrument: constant pressure and constant temperature scald instrument

[0106] Thirty-two healthy rats, weighing no more than 20 grams, were randomly divided into eight groups. A deep second-degree burn model was created using a constant-pressure, constant-temperature burn apparatus. After 24 hours of observation, significant inflammatory changes, such as purple-red hyperemia and edema, were observed on the wound surface. The burn area and depth were similar across all groups, and the wound surface area was recorded.

[0107] After the animal model was established, each group was housed in a cage. The wounds of the rats in the blank group were not treated. The wounds of the rats in the experimental and control groups were smeared with 0.5 ml of cell regeneration agent every day for 14 consecutive days. The growth of the wounds of the rats in each group was recorded, and the wound recovery rate of each group was calculated. The results are as follows: Figure 2 shown.

[0108] The calculation formula of recovery rate is: recovery rate = (S0-S t ) / S0, where S0 is the average initial area of ​​the rat wounds and St is the average area of ​​the rat wounds on day t.

[0109] from Figure 2 It can be seen from the results of the blank group and the three groups of Examples 5 to 6 that the wound recovery rates of the two groups of Examples 5 to 6 are significantly higher, indicating that the cell regeneration agent obtained by adding L-tryptophanol oxalate and isosorbide-2-acetate to the F12 complete medium is more conducive to the recovery of rat wounds, and further indicating that the addition of L-tryptophanol oxalate and isosorbide-2-acetate to the F12 complete medium is beneficial to promoting cell regeneration; at the same time, the higher the amount of L-tryptophanol oxalate and isosorbide-2-acetate is, the more conducive it is to promoting cell regeneration; the comparison of the results of the three groups of Examples 6 to 8, Example 7 The wound recovery rates of the two groups were significantly higher, indicating that the cell regeneration agent obtained by adding licorice alcohol to the F12 complete culture medium is more conducive to the recovery of rat wounds, and further indicates that the addition of licorice alcohol to the F12 complete culture medium is beneficial to promoting cell regeneration; at the same time, the higher the dosage of licorice alcohol, the more conducive it is to promoting cell regeneration; comparing the results of the blank group and the comparative examples 1~2, there was no significant improvement in the wound recovery rates of the two groups of comparative examples 1~2, indicating that the cell regeneration agent obtained by adding only L-tryptophanol oxalate or isosorbide-2-acetate to the F12 complete culture medium is not conducive to the recovery of rat wounds.

[0110] Experimental Example 3

[0111] Cells: Umbilical cord mesenchymal stem cells

[0112] Experimental group: cell models of Examples 9-13

[0113] Control group: cell model of comparative example 3-4

[0114] Exosomes are tiny vesicles secreted by cells. Exosomes are primarily derived from multivesicular bodies formed by the invagination of lysosomal microparticles within cells. Exosomes are composed of a lipid bilayer and contain all known molecular components of cells, including proteins, nucleic acids, and cholesterol. Exosomes play a variety of important roles in the body, including cell communication and signaling, immune regulation, tissue repair and regeneration, drug delivery systems, and diagnosis and monitoring. Among them, the surface markers of exosomes mainly include CD63, CD81, CD9, TSG101, and HSP27. In the present invention, the amount of exosomes is characterized by the detection results of CD63.

[0115] Western Blot assay was used to detect the expression of exosome marker protein CD63. Figure 3 shown.

[0116] from Figure 3 It can be seen that, by comparing the results of the three groups of Examples 9 to 11, the CD63 expression levels of the two groups of Examples 10 and 11 were higher, indicating that the addition of L-tryptophanol oxalate and isosorbide-2-acetate to the F12 complete medium can effectively increase the expression level of CD63 in the cells, and further indicating that the addition of L-tryptophanol oxalate and isosorbide-2-acetate to the F12 complete medium can effectively increase the amount of exosomes in the umbilical cord mesenchymal stem cells; by comparing the results of the three groups of Examples 11 to 13, the CD63 expression levels of the two groups of Examples 12 and 13 were higher, indicating that the addition of glycyrrhizin to the F12 complete medium can effectively increase the amount of exosomes in the umbilical cord mesenchymal stem cells. The expression level of CD63 in the umbilical cord mesenchymal stem cells was increased, which further indicated that the addition of L-tryptophanol oxalate and isosorbide-2-acetate to the F12 complete medium can effectively increase the amount of exosomes in umbilical cord mesenchymal stem cells; comparing the three groups of data results of Example 9 and Comparative Examples 3~4, the CD63 expression levels in the two groups of results of Comparative Examples 3~4 did not increase significantly, indicating that the addition of L-tryptophanol oxalate or isosorbide-2-acetate alone to the F12 complete medium is not conducive to increasing the expression level of CD63 in umbilical cord mesenchymal stem cells, which further indicated that the addition of L-tryptophanol oxalate or isosorbide-2-acetate alone to the F12 complete medium is not conducive to increasing the amount of exosomes in umbilical cord mesenchymal stem cells.

[0117] The embodiments and / or implementation methods described above are only used to illustrate the preferred embodiments and / or implementation methods for realizing the technology of the present invention, and do not impose any form of limitation on the implementation methods of the technology of the present invention. Any person skilled in the art may make slight changes or modifications to other equivalent embodiments without departing from the scope of the technical means disclosed in the content of the present invention, but they should still be regarded as technologies or embodiments that are essentially the same as the present invention.

[0118] This article uses specific examples to illustrate the principles and implementation methods of this application. The description of the above embodiments is only used to help understand the method and core ideas of this application. The above is only the preferred implementation method of this application. It should be pointed out that due to the limitations of textual expression, there are objectively infinite specific structures. For ordinary technicians in this technical field, without departing from the principles of this application, they can also make several improvements, modifications or changes, and can also combine the above technical features in an appropriate manner; these improvements, modifications, changes or combinations, or the direct application of the inventive concept and technical solution to other occasions without improvement, should be regarded as the scope of protection of this application.

Claims

1. A cell regeneration agent, comprising L-tryptophanol oxalate and isosorbide-2-acetate, wherein the L-tryptophanol oxalate and isosorbide-2-acetate are mixed in a mass ratio of 1:1 to 4, and the cells are umbilical cord mesenchymal stem cells.

2. A cell regeneration agent according to claim 1, characterized in that The cell regeneration agent also includes F12 complete culture medium, and the concentration of the composition in the F12 complete culture medium is 0.002-0.008 g / ml.

3. A method for constructing a cell model, comprising: Cells are cultured using the cell regeneration agent according to any one of claims 1-2 to obtain a cell model, wherein the cells are umbilical cord mesenchymal stem cells.

4. The method for constructing a cell model according to claim 3, wherein: The exosomes secreted by the cells in the cell model contain at least one of nucleic acid, protein and cholesterol.

5. The method for constructing a cell model according to claim 4, characterized in that: The surface markers of the exosomes include at least one of CD63, CD81, CD9, TSG101 and HSP27.

6. Use of the cell regeneration agent according to claim 1 in preparing a cell model, wherein the cells are umbilical cord mesenchymal stem cells.

7. Use of the cell regeneration agent according to claim 1 in the preparation of wound repair materials, wherein the cells are umbilical cord mesenchymal stem cells.

8. Use of the cell regeneration agent according to claim 1 in the preparation of a wound recovery agent, wherein the cells are umbilical cord mesenchymal stem cells.

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