A composition containing mesenchymal stem cells and its use in the preparation of products with anti-inflammatory action

Abstract

The application belongs to the technical field of stem cells, and specifically discloses a mesenchymal stem cell-containing composition and application of the mesenchymal stem cell-containing composition in preparation of a product with an anti-inflammatory effect. The lycopene and oleuropein are added to an activation culture medium. On one hand, the lycopene and the oleuropein can protect the biological activity of the mesenchymal stem cells, improve the immune regulation function of the mesenchymal stem cells, further promote the repair of tissues, improve the proliferation capacity of the mesenchymal stem cells, and promote the generation and secretion of active factors with an anti-inflammatory effect in the mesenchymal stem cells and effectively protect the activity of the active factors. On the other hand, the synergistic effect of the lycopene and the oleuropein can more effectively reduce the tissue damage caused by oxidative stress and inflammation, and significantly improve the anti-inflammatory effect of the composition.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of stem cells, in particular to a mesenchymal stem cell-containing composition and application thereof in preparing a product with anti-inflammatory effect. BACKGROUND

[0002] Mesenchymal stem cells (MSCs) are a kind of adult stem cells with high proliferative and multi-differentiation potential, which are widely used in tissue repair and regenerative medicine. Mesenchymal stem cells have self-renewal and multi-differentiation ability, and also exhibit strong immunomodulatory and anti-inflammatory effects, and are widely used in the treatment of various inflammatory diseases.

[0003] Mesenchymal stem cells are susceptible to oxidative stress and culture medium when cultured in vitro and applied in vivo, resulting in reduced survival rate and weakened secretion function, which limits their anti-inflammatory effect. In the prior art, the function of MSCs can be partially improved by adding growth factors (such as EGF) or antioxidants (such as glutathione), but single component cannot simultaneously solve the problems of poor anti-inflammatory effect. SUMMARY

[0004] The present application provides a mesenchymal stem cell-containing composition and application thereof in preparing a product with anti-inflammatory effect. The mesenchymal stem cell-containing composition has excellent anti-inflammatory effect.

[0005] The present application solves the technical problems by adopting the following technical solutions:

[0006] A preparation method of a mesenchymal stem cell-containing composition, comprising the following steps:

[0007] Mesenchymal stem cells are inoculated in a basic culture medium, and when the cell confluence reaches 78-82%, the culture medium is replaced with an activation culture medium, and the culture is continued for 24-72 hours, followed by centrifugation, filtration, and obtaining of the mesenchymal stem cell-containing composition;

[0008] The activation culture medium comprises the following components: 8-12% fetal bovine serum, 1-5 g / L lycopene, 1-5 g / L oleuropein, 50-80 μg / mL glutathione, 0.02-0.05 mM aspartic acid, 20-60 ng / mL epidermal growth factor, 0.2-0.6 mg / L D-galactose, 50-120 μg / L linolenic acid, and the balance of DMEM culture medium.

[0009] Lycopene: It can effectively scavenge free radicals (such as superoxide anion, hydrogen peroxide, etc.), reduce oxidative stress. Free radicals and oxidative stress are one of the key factors that trigger inflammatory response, therefore, lycopene can inhibit inflammation triggered by oxidative damage through antioxidant effect, at the same time, lycopene can reduce the secretion of pro-inflammatory cytokines (such as TNF-α, IL-1β, IL-6, etc.) by regulating inflammation-related pathways (such as NF-κB, MAPK, etc.). This helps to reduce inflammatory response, thereby improving tissue repair process.

[0010] Oleuropein: Oleuropein has strong antioxidant capacity, can neutralize free radicals and reduce oxidative damage. It can enhance the activity of intracellular antioxidant enzymes (such as superoxide dismutase, catalase, etc.), promote the improvement of cell antioxidant capacity, at the same time, it has excellent anti-inflammatory effect, can reduce the tissue damage caused by chronic inflammation, and is helpful to improve the efficiency of tissue repair.

[0011] The present application creatively adds lycopene and oleuropein to the activation medium. On the one hand, lycopene and oleuropein can protect the biological activity of mesenchymal stem cells, improve the immune regulation function of mesenchymal stem cells, and then promote the repair of tissues, improve the proliferation ability of mesenchymal stem cells, and promote the production and secretion of active factors with anti-inflammatory effect in mesenchymal stem cells and effectively protect their activity; on the other hand, the synergistic effect of the two can more effectively reduce the tissue damage caused by oxidative stress and inflammation, and significantly improve the anti-inflammatory effect of the composition.

[0012] As a preferred embodiment of the present application, the activation medium comprises the following components: 8-12% fetal bovine serum, 2-4g / L lycopene, 2-4g / L oleuropein, 50-80μg / mL glutathione, 0.02-0.05mM aspartic acid, 20-60ng / mL epidermal growth factor, 0.2-0.6mg / L D-galactose, 50-120μg / L linolenic acid, and the balance of DMEM medium. Especially when the amount of each raw material is within this range, the anti-inflammatory effect can be more significantly improved.

[0013] As a preferred embodiment of the present application, the basic medium is DMEM medium.

[0014] As a preferred embodiment of the present application, the mesenchymal stem cell inoculation density is 0.5-4×10 6 cells / mL.

[0015] As a preferred embodiment of the present application, the mesenchymal stem cell inoculation density is 1-2×10 6 cells / mL.

[0016] As a preferred embodiment of the present application, the culture is carried out in a 36-38℃ incubator with 5% CO2.

[0017] As a preferred embodiment of the present application, the culture is carried out in a 37℃ incubator with 5% CO2.

[0018] As a preferred embodiment of the present application, the mesenchymal stem cells are human placental mesenchymal stem cells.

[0019] The present application also provides a mesenchymal stem cell-containing composition prepared by the preparation method described above.

[0020] The present application also provides the use of a mesenchymal stem cell-containing composition in the preparation of a product with anti-inflammatory effect.

[0021] The present application has the following beneficial effects: by adding the lycopene and oleuropein to the activation culture medium, on the one hand, the lycopene and oleuropein can protect the biological activity of the mesenchymal stem cells, improve the immune regulation function of the mesenchymal stem cells, and further promote the repair of tissues and improve the proliferation ability of the mesenchymal stem cells, and promote the production and secretion of active factors with anti-inflammatory effect in the mesenchymal stem cells and effectively protect the activity thereof; on the other hand, the synergistic effect of the two can more effectively reduce the tissue damage caused by oxidative stress and inflammation, and significantly improve the anti-inflammatory effect of the composition. DETAILED DESCRIPTION

[0022] To make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments are some but not all of the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0023] In the present application, the technical features described in an open way include both the closed technical solutions consisting of the listed features and the open technical solutions containing the listed features.

[0024] In the present application, as long as there is no special description, the numerical range is regarded as continuous and includes the minimum value and the maximum value of the range and each value between the minimum value and the maximum value. Further, when the range refers to an integer, each integer between the minimum value and the maximum value of the range is included. In addition, when multiple ranges are provided to describe a feature or a characteristic, the ranges can be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all sub-ranges subsumed therein.

[0025] In the present application, the specific dispersion, stirring treatment method is not particularly limited.

[0026] The reagents or instruments used in the present application are not specified by the manufacturer, and are all conventional products that can be obtained by purchase.

[0027] Example 1

[0028] A method for preparing a mesenchymal stem cell-containing composition, comprising the following steps:

[0029] Human placental mesenchymal stem cells are inoculated in DMEM medium at 0.5 x 10 6 cells / mL, and when the cell confluence reaches 80%, the medium is replaced with an activation medium, and the culture is continued for 54 h, followed by centrifugation and filtration to obtain the mesenchymal stem cell-containing composition. The culture is carried out in a 37°C, 5% CO2 incubator.

[0030] The activation medium comprises the following components: fetal bovine serum 10%, lycopene 2 g / L, oleuropein 4 g / L, glutathione 60 μg / mL, aspartic acid 0.04 mM, epidermal growth factor 40 ng / mL, D-galactose 0.5 mg / L, linolenic acid 100 μg / L, and the balance DMEM medium.

[0031] The human placental mesenchymal stem cells are derived from Shanghai Zhen Industry, and the model number is HZH-5204.

[0032] Example 2

[0033] A method for preparing a mesenchymal stem cell-containing composition, comprising the following steps:

[0034] Human placental mesenchymal stem cells are inoculated in DMEM medium at 0.5 x 10 6 cells / mL, and when the cell confluence reaches 80%, the medium is replaced with an activation medium, and the culture is continued for 54 h, followed by centrifugation and filtration to obtain the mesenchymal stem cell-containing composition. The culture is carried out in a 37°C, 5% CO2 incubator.

[0035] The activation medium comprises the following components: fetal bovine serum 10%, lycopene 4 g / L, oleuropein 2 g / L, glutathione 60 μg / mL, aspartic acid 0.04 mM, epidermal growth factor 40 ng / mL, D-galactose 0.5 mg / L, linolenic acid 100 μg / L, and the balance DMEM medium.

[0036] The human placental mesenchymal stem cells are derived from Shanghai Zhen Industry, and the model number is HZH-5204.

[0037] Example 3

[0038] A method for preparing a mesenchymal stem cell-containing composition, comprising the following steps:

[0039] Human placental mesenchymal stem cells are inoculated in DMEM medium at 0.5 x 10 6 cells / mL, and when the cell confluence reaches 80%, the medium is replaced with an activation medium, and the culture is continued for 54 h, followed by centrifugation and filtration to obtain the mesenchymal stem cell-containing composition. The culture is carried out in a 37℃, 5% CO2 incubator.

[0040] The activation medium comprises the following components: fetal bovine serum 10%, lycopene 1 g / L, oleuropein 5 g / L, glutathione 60 μg / mL, aspartic acid 0.04 mM, epidermal growth factor 40 ng / mL, D-galactose 0.5 mg / L, linolenic acid 100 μg / L, and the balance DMEM medium.

[0041] The human placental mesenchymal stem cells are from Shanghai Zhen Industry, and the model number is HZH-5204.

[0042] Example 4

[0043] A method for preparing a mesenchymal stem cell-containing composition, comprising the following steps:

[0044] Human placental mesenchymal stem cells are inoculated in DMEM medium at 0.5 x 10 6 cells / mL, and when the cell confluence reaches 80%, the medium is replaced with an activation medium, and the culture is continued for 54 h, followed by centrifugation and filtration to obtain the mesenchymal stem cell-containing composition. The culture is carried out in a 37℃, 5% CO2 incubator.

[0045] The activation medium comprises the following components: fetal bovine serum 10%, lycopene 1 g / L, oleuropein 5 g / L, glutathione 60 μg / mL, aspartic acid 0.04 mM, epidermal growth factor 40 ng / mL, D-galactose 0.5 mg / L, linolenic acid 100 μg / L, and the balance DMEM medium.

[0046] The human placental mesenchymal stem cells are from Shanghai Zhen Industry, and the model number is HZH-5204.

[0047] Example 5

[0048] A method for preparing a mesenchymal stem cell-containing composition, comprising the following steps:

[0049] Human placental mesenchymal stem cells were used at a concentration of 0.5 × 10⁻⁶. 6 Cells were seeded at a density of 1 / mL in DMEM medium and cultured until 80% confluence was achieved. The culture medium was then replaced with activation medium, and the cells were cultured for another 54 hours. After centrifugation and filtration, a composition containing mesenchymal stem cells was obtained. The culture was conducted in an incubator at 37°C and 5% CO2.

[0050] The activation medium comprises the following components: 10% fetal bovine serum, 2 g / L lycopene, 4 g / L oleuropein, 50 μg / mL glutathione, 0.05 mM aspartic acid, 60 ng / mL epidermal growth factor, 0.2 mg / L D-galactose, 120 μg / L linolenic acid, and the remainder being DMEM medium.

[0051] The human placental mesenchymal stem cells mentioned above are from Huzhen Industrial Co., Ltd., brand name: HZH-5204.

[0052] Example 6

[0053] A method for preparing a composition containing mesenchymal stem cells includes the following steps:

[0054] Human placental mesenchymal stem cells were used at a concentration of 0.5 × 10⁻⁶. 6 Cells were seeded at a density of 1 / mL in DMEM medium and cultured until 80% confluence was achieved. The culture medium was then replaced with activation medium, and the cells were cultured for another 54 hours. After centrifugation and filtration, a composition containing mesenchymal stem cells was obtained. The culture was conducted in an incubator at 37°C and 5% CO2.

[0055] The activation medium comprises the following components: 10% fetal bovine serum, 2 g / L lycopene, 4 g / L oleuropein, 80 μg / mL glutathione, 0.02 mM aspartic acid, 20 ng / mL epidermal growth factor, 0.6 mg / L D-galactose, 50 μg / L linolenic acid, and the remainder being DMEM medium.

[0056] The human placental mesenchymal stem cells mentioned above are from Huzhen Industrial Co., Ltd., brand name: HZH-5204.

[0057] Comparative Example 1

[0058] The difference between Comparative Example 1 and Example 1 is that the activation culture medium of Comparative Example 1 does not contain lycopene, but all other aspects are the same.

[0059] The activation medium comprises the following components: 10% fetal bovine serum, 4 g / L oleuropein, 60 μg / mL glutathione, 0.04 mM aspartic acid, 40 ng / mL epidermal growth factor, 0.5 mg / L D-galactose, 100 μg / L linolenic acid, and the remainder being DMEM medium.

[0060] Comparative Example 2

[0061] The difference between Comparative Example 2 and Example 1 is that the activation culture medium of Comparative Example 2 does not contain oleuropein, but all other aspects are the same.

[0062] The activation medium comprises the following components: 10% fetal bovine serum, 2 g / L lycopene, 60 μg / mL glutathione, 0.04 mM aspartic acid, 40 ng / mL epidermal growth factor, 0.5 mg / L D-galactose, 100 μg / L linolenic acid, and the remainder being DMEM medium.

[0063] Comparative Example 3

[0064] The difference between Comparative Example 3 and Example 1 is that the activation culture medium of Comparative Example 3 does not contain lycopene (the total amount of lycopene and oleuropein remains unchanged), while everything else is the same.

[0065] The activation medium comprises the following components: 10% fetal bovine serum, 6 g / L oleuropein, 60 μg / mL glutathione, 0.04 mM aspartic acid, 40 ng / mL epidermal growth factor, 0.5 mg / L D-galactose, 100 μg / L linolenic acid, and the remainder being DMEM medium.

[0066] Comparative Example 4

[0067] The difference between Comparative Example 4 and Example 1 is that the activation culture medium of Comparative Example 4 does not contain oleuropein (the total amount of lycopene and oleuropein remains unchanged), while everything else is the same.

[0068] The activation medium comprises the following components: 10% fetal bovine serum, 6 g / L lycopene, 60 μg / mL glutathione, 0.04 mM aspartic acid, 40 ng / mL epidermal growth factor, 0.5 mg / L D-galactose, 100 μg / L linolenic acid, and the remainder being DMEM medium.

[0069] Comparative Example 5

[0070] The difference between Comparative Example 5 and Example 1 is that Comparative Example 5 uses an equal amount of sea buckthorn flavonoids to replace lycopene, while everything else is the same.

[0071] The activation medium comprises the following components: 10% fetal bovine serum, 2 g / L sea buckthorn flavonoids, 4 g / L oleuropein, 60 μg / mL glutathione, 0.04 mM aspartic acid, 40 ng / mL epidermal growth factor, 0.5 mg / L D-galactose, 100 μg / L linolenic acid, and the remainder being DMEM medium.

[0072] Comparative Example 6

[0073] The difference between Comparative Example 6 and Example 1 is that Comparative Example 6 uses an equal amount of okra lactone to replace oleuropein, while all other aspects are the same.

[0074] The activation medium comprises the following components: 10% fetal bovine serum, 2 g / L lycopene, 4 g / L omega-3 lactone, 60 μg / mL glutathione, 0.04 mM aspartic acid, 40 ng / mL epidermal growth factor, 0.5 mg / L D-galactose, 100 μg / L linolenic acid, and the remainder being DMEM medium.

[0075] Comparative Example 7

[0076] The difference between Comparative Example 7 and Example 1 is that the amounts of lycopene and oleuropein are not within the scope of this invention, but everything else is the same.

[0077] The activation medium comprises the following components: 10% fetal bovine serum, 0.5 g / L lycopene, 5 g / L oleuropein, 60 μg / mL glutathione, 0.04 mM aspartic acid, 40 ng / mL epidermal growth factor, 0.5 mg / L D-galactose, 100 μg / L linolenic acid, and the remainder being DMEM medium.

[0078] Comparative Example 8

[0079] The difference between Comparative Example 8 and Example 1 is that the amounts of lycopene and oleuropein are not within the scope of this invention, but everything else is the same.

[0080] The activation medium comprises the following components: 10% fetal bovine serum, 5.5 g / L lycopene, 0.5 g / L oleuropein, 60 μg / mL glutathione, 0.04 mM aspartic acid, 40 ng / mL epidermal growth factor, 0.5 mg / L D-galactose, 100 μg / L linolenic acid, and the remainder being DMEM medium.

[0081] Test case

[0082] HaCaT (human keratinocytes) were cultured in Medium 154 medium containing 10% fetal bovine serum, 100 U / ml penicillin, and 100 μg / ml streptomycin at 37°C, 5% CO2, and 98% humidity. Logarithmic growth phase cells were selected and seeded in 96-well plates. After 24 hours of complete cell adhesion, the medium was replaced with Medium 154 medium containing the samples from the examples and comparative examples (final concentration 0.1 mg / mL) (the negative control group was not replaced). After 12 hours of incubation, the medium was increased to 25 mJ / cm². 2 The cells were irradiated with UVB for 10 minutes. After irradiation, the cell culture supernatant was collected, and the concentrations of IL-1α and IL-16 in the supernatant were detected using a human ELISA kit. Each experiment was repeated three times, and the average value was taken as the final result. The results are shown in Table 1.

[0083] IL-1α inhibition rate (%) = (IL-1α expression level in sample / IL-1α expression level in negative control group) × 100%.

[0084] IL-6 inhibition rate (%) = (IL-6 expression level in sample / IL-6 expression level in negative control group) × 100%.

[0085] Table 1

[0086]

[0087] As can be seen from Table 1, the composition containing mesenchymal stem cells described in this invention has excellent anti-inflammatory effects.

[0088] The experimental data show that the activation culture medium is the key to the excellent anti-inflammatory effect of the composition containing mesenchymal stem cells of the present invention. By using the specific activation culture medium of the present invention, the anti-inflammatory effect of the composition containing mesenchymal stem cells can be significantly improved.

[0089] Comparing Example 1 with Comparative Examples 1-6, it can be seen that the lycopene and oleuropein described in this invention have a synergistic effect. The absence of either one, or the substitution of either one with other components, will lead to a significant decrease in the anti-inflammatory effect.

[0090] Finally, it should be noted that the above embodiments are used to illustrate the technical solutions of the present invention and not to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for preparing a composition containing mesenchymal stem cells, characterized in that, Includes the following steps: Mesenchymal stem cells were seeded in basal culture medium and cultured until the cell confluence reached 78-82%. Then, the medium was replaced with activation culture medium and cultured for another 24-72 hours. The cells were then centrifuged and filtered to obtain a composition containing mesenchymal stem cells. The activation medium consists of the following components: 8-12% fetal bovine serum, 1-5 g / L lycopene, 1-5 g / L oleuropein, 50-80 μg / mL glutathione, 0.02-0.05 mM aspartic acid, 20-60 ng / mL epidermal growth factor, 0.2-0.6 mg / L D-galactose, 50-120 μg / L linolenic acid, and the balance being DMEM medium.

2. The method for preparing the composition containing mesenchymal stem cells according to claim 1, characterized in that, The activation medium consists of the following components: 8-12% fetal bovine serum, 2-4 g / L lycopene, 2-4 g / L oleuropein, 50-80 μg / mL glutathione, 0.02-0.05 mM aspartic acid, 20-60 ng / mL epidermal growth factor, 0.2-0.6 mg / L D-galactose, 50-120 μg / L linolenic acid, and the remainder being DMEM medium.

3. The method for preparing the composition containing mesenchymal stem cells according to claim 1, characterized in that, The basal culture medium is DMEM medium.

4. The method for preparing the composition containing mesenchymal stem cells according to claim 1, characterized in that, The mesenchymal stem cell seeding density is (0.5~4)×10⁻⁶. 6 per mL.

5. The method for preparing the composition containing mesenchymal stem cells according to claim 1, characterized in that, The culture was carried out in an incubator at 36-38℃ and 5% CO2.

6. The method for preparing the composition containing mesenchymal stem cells according to claim 1, characterized in that, The culture was carried out in an incubator at 37°C and 5% CO2.

7. The method for preparing the composition containing mesenchymal stem cells according to claim 1, characterized in that, The mesenchymal stem cells mentioned are human placental mesenchymal stem cells.

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