An induction medium for increasing the IL-10 secretion amount of mesenchymal stem cells and its application
By adding IFN-γ mimicking peptide and TNF-ɑ mimicking peptide to mesenchymal stem cell culture medium, the problem of increasing IL-10 secretion in the prior art affecting cell proliferation is solved, and a significant increase in IL-10 secretion is achieved. It is suitable for the preparation of cellular drugs for the treatment of inflammatory diseases.
Patent Information
- Application Number
- CN202411913088.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-24
- Publication Date
- 2025-08-01
- Estimated Expiration
- 2044-12-24
AI Technical Summary
In the prior art methods to increase the secretion amount of IL-10 in mesenchymal stem cells, there is a problem of complex genetic engineering and high cost that affects cell proliferation. It is difficult to increase the secretion amount of IL-10 without affecting the proliferation ability of cells.
The amplified human mesenchymal cells were induced and cultured using basal culture medium containing human IFN-γ mimic peptide and TNF-ɑ mimic peptide. By adding a specific amount of IFN-γ mimic peptide and TNF-ɑ mimic peptide to the basal culture medium, cells were harvested after 12-24 hours of induction and culture, and mesenchymal stem cells with increased secretion of IL-10 were obtained.
The secretion of IL-10 is significantly increased without affecting the proliferation ability of mesenchymal stem cells, providing cellular drugs for the preparation of inflammatory diseases.
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Figure CN119464203B_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of stem cell culture, and particularly relates to an induction medium for increasing the secretion amount of interleukin-10 of mesenchymal stem cells and its application. Background Art
[0002] Mesenchymal stem cells (MSCs) are important members of the stem cell family. They are a type of adult stem cells with self-renewal ability and multi-directional differentiation potential. They are derived from the mesoderm in the early stage of development and mainly exist in bone marrow, adipose tissue, umbilical cord blood, umbilical cord tissue and other parts. MSCs were initially discovered in bone marrow and have attracted increasing attention due to their characteristics such as multi-directional differentiation potential, hematopoietic support and promotion of stem cell implantation, immune regulation and self-replication.
[0003] Among them, MSCs have important application potential in immune regulation because they can inhibit immune responses through various mechanisms, thus showing potential therapeutic effects in the treatment of autoimmune diseases, organ transplantation rejection reactions and inflammatory diseases. For example, MSCs can inhibit the proliferation and activation of T cells by secreting soluble factors (such as transforming growth factor-β, interleukin-10, etc.) or direct cell-cell contact. The secretion of interleukin-10 (IL-10) by MSCs is an important aspect of their immune regulation function. IL-10 is a powerful anti-inflammatory factor that can inhibit the activation and proliferation of various immune cells, thereby reducing the inflammatory response.
[0004] However, there are also some potential drawbacks and challenges in the process of MSCs secreting IL-10. Under in vitro culture conditions, MSCs usually secrete a certain amount of IL-10, and the secretion level of IL-10 can vary depending on the source of MSCs, culture conditions, cell status, and the presence of stimulating factors (such as inflammatory mediators, cytokines, etc.). Under normal circumstances, the level of IL-10 secreted by MSCs is relatively low, but when MSCs encounter inflammatory signals or immune cells, they may increase the secretion of IL-10 to exert their immunomodulatory effects. This increase may be due to MSCs responding to signals in the surrounding environment, such as inflammatory factors like TNF-α and IFN-γ, which can induce MSCs to secrete more IL-10 and other anti-inflammatory factors. In practical applications, researchers may use different methods to enhance the ability of MSCs to secrete IL-10, such as enhancing the expression of IL-10 through genetic engineering means, or inducing MSCs to secrete more IL-10 through specific culture conditions. However, the process of genetic engineering means is complex, involves a large number of exogenous substances, is difficult to control, and there are doubts about its clinical application safety; the method of inducing MSCs to secrete more IL-10 through specific culture conditions, such as adding TNF-α, IFN-γ, etc. in the culture medium, has a high production cost of protein factors (the selling price per milligram is generally over a thousand yuan, directly affecting the cost of cell preparation), and the stimulation of protein factors will, to a certain extent, affect the proliferation ability of MSCs and promote cell senescence and other problems. Therefore, providing a method to increase the IL-10 secretion of mesenchymal stem cells, while not affecting the proliferation ability of MSCs, is an urgent problem to be solved. Summary of the Invention
[0005] In order to provide a method that can both increase the IL-10 secretion of mesenchymal stem cells and not affect the proliferation of mesenchymal stem cells, the present invention uses a basal medium containing human IFN-γ mimetic peptide and TNF-α mimetic peptide to induce the amplified human mesenchymal cells, and then obtains mesenchymal stem cells with increased IL-10 secretion after harvest culture. The induction medium provided by the present invention can increase the IL-10 secretion of mesenchymal stem cells while not affecting the proliferation of mesenchymal stem cells, and the induced mesenchymal stem cells are expected to be applied to the preparation of cell drugs for treating inflammatory diseases.
[0006] To solve the above technical problems and achieve the corresponding technical effects, the present invention provides the following technical solutions:
[0007] The first object of the present invention is to provide an induction medium for increasing the IL-10 secretion of mesenchymal stem cells. The induction medium is obtained by adding cytokines to a basal medium. The cytokines are a human IFN-γ mimetic peptide and a TNF-α mimetic peptide. The amino acid sequence of the human IFN-γ mimetic peptide is as shown in SEQ ID NO.1, and the corresponding amino acid sequence of the TNF-α mimetic peptide is NKHNRKI.
[0008] In one embodiment of the present invention, the addition amount of the human IFN-γ mimetic peptide is 200 - 1000 U / mL, and the addition amount of the TNF-α mimetic peptide is 5 - 10 ng / mL.
[0009] In one embodiment of the present invention, the addition amount of the human IFN-γ mimetic peptide is 600 U / mL, and the addition amount of the TNF-α mimetic peptide is 10 ng / mL.
[0010] In one embodiment of the present invention, the basal medium is an α-MEM medium containing double antibiotics and 10 - 20% FBS or double antibiotics and 10 - 20% human AB serum.
[0011] The second object of the present invention is to provide the application of the above induction medium in increasing the IL-10 secretion of mesenchymal stem cells.
[0012] The third object of the present invention is to provide a method for increasing the IL-10 secretion of mesenchymal stem cells. The method comprises the following steps:
[0013] S1. Amplification culture: Amplify and culture to obtain a sufficient amount of human mesenchymal stem cells as the first mesenchymal stem cells.
[0014] S2. Induction culture: Place the first mesenchymal stem cells obtained in S1 in the above induction medium, and culture them under the conditions of 2 - 5% O2, 5% CO2, and 37°C for 12 - 24 h to obtain the second mesenchymal stem cells.
[0015] S3. Harvest culture: Discard the culture solution obtained in the culture of S2, wash the cells twice with physiological saline, digest the cells with trypsin and harvest the cells, and then inoculate them into an ultra-low adsorption 96-well plate at an inoculation amount of 1×10 6 cells / mL, centrifuge, add an α-MEM medium containing 5 - 10% human AB serum, and culture them under the conditions of 5% O2, 5% CO2, and 37°C for 12 h.
[0016] In one embodiment of the present invention, for the amplification culture in S1, human mesenchymal stem cells are resuscitated in a 37°C water bath and then inoculated into a basal medium, and cultured under the conditions of 5% O2, 5% CO2, and 37°C until the cell confluence reaches 70%-90%; the basal medium is α-MEM medium containing double antibodies and 10-20% FBS or double antibodies and 10-20% human AB serum.
[0017] In one embodiment of the present invention, the human mesenchymal stem cells in S1 are human bone marrow mesenchymal stem cells, human umbilical cord mesenchymal stem cells, or human placental chorionic mesenchymal stem cells.
[0018] The fourth object of the present invention is to provide human mesenchymal stem cells obtained by the above method.
[0019] The fifth object of the present invention is to provide the application of the above human mesenchymal stem cells in the preparation of cell drugs for treating inflammatory diseases.
[0020] Advantages of the present invention:
[0021] In the present invention, human mesenchymal cells after amplification are induced with a basal medium containing human IFN-γ mimetic peptide and TNF-α mimetic peptide, and then human mesenchymal stem cells with increased IL-10 secretion are obtained after harvest culture; through the detection of the proliferation ability of mesenchymal stem cells, it is found that the induction medium provided by the present invention can increase the IL-10 secretion of mesenchymal stem cells without affecting the proliferation of mesenchymal stem cells, and the induced mesenchymal stem cells are expected to be applied to the preparation of cell drugs for treating inflammatory diseases. Description of the drawings
[0022] Figure 1 It is a BCA method detection result graph of the total protein concentration in the supernatant after lysis of human umbilical cord mesenchymal stem cells obtained in Examples 4-6; among them, hypoxia-, represents a normoxic culture environment of 20% O2, hypoxia+, represents a hypoxic culture environment of 5% O2, and ①②③ respectively represent adding human IFN-γ mimetic peptide and TNF-α mimetic peptide according to the addition amounts in Examples 1, 2, and 3.
[0023] Figure 2 It is an ELISA detection result graph of the concentration of anti-inflammatory factor IL-10 in the supernatant after lysis of human umbilical cord mesenchymal stem cells obtained in Examples 4-6; among them, hypoxia-, represents a normoxic culture environment of 20% O2, hypoxia+, represents a hypoxic culture environment of 5% O2, and ①②③ respectively represent adding human IFN-γ mimetic peptide and TNF-α mimetic peptide according to the addition amounts in Examples 1, 2, and 3.
[0024] Figure 3Detection result graph of the proliferation ability of human umbilical cord mesenchymal stem cells cultured in the induction medium described in Example 1. Detailed implementation manners
[0025] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below in combination with specific implementation manners and the attached drawings of the specification. The embodiments described in the present invention are not all embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present invention without creative efforts fall within the scope of protection of the present invention.
[0026] In the ranges disclosed in the invention, the endpoints and any values of the ranges are not limited to the exact ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, between the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values, they can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.
[0027] The experimental methods used in the following embodiments are all conventional methods unless otherwise specified, and the materials, reagents and instruments used, unless otherwise specified, are all conventional materials, reagents and instruments in the art, and those skilled in the art can obtain them through commercial channels.
[0028] The amino acid sequence of the human IFN-γ mimetic peptide used in the present invention is as shown in SEQ ID NO.1; the amino acid sequence corresponding to the TNF-α mimetic peptide used in the present invention is NKHNRKI.
[0029] SEQ ID NO.1: LTNYSVTDLNVQRKAIHELIQVMAELSPAAKTGKRKRSQM
[0030] Example 1: An induction medium for increasing the IL-10 secretion amount of mesenchymal stem cells
[0031] The induction medium described in this example is prepared by adding a human IFN-γ mimetic peptide and a TNF-α mimetic peptide to a basal medium. The specific composition of this induction medium is α-MEM medium, double antibodies, 10% FBS, 300 U / mL of human IFN-γ mimetic peptide, and 10 ng / mL of TNF-α mimetic peptide.
[0032] Example 2: An induction medium for increasing the IL-10 secretion amount of mesenchymal stem cells
[0033] The induction medium described in this example is prepared by adding human IFN-γ mimetic peptide and TNF-α mimetic peptide to a basal medium. The specific composition of this induction medium is α-MEM medium, double antibody, 10% FBS, 1000 U / mL of human IFN-γ mimetic peptide, and 5 ng / mL of TNF-α mimetic peptide.
[0034] Example 3: An induction medium for increasing the IL-10 secretion of mesenchymal stem cells
[0035] The induction medium described in this example is prepared by adding human IFN-γ mimetic peptide and TNF-α mimetic peptide to a basal medium. The specific composition of this induction medium is α-MEM medium, double antibody, 10% FBS, 600 U / mL of human IFN-γ mimetic peptide, and 10 ng / mL of TNF-α mimetic peptide.
[0036] Example 4: A method for increasing the IL-10 secretion of mesenchymal stem cells
[0037] S1. Amplification culture: Resuscitate human umbilical cord mesenchymal stem cells in a 37°C water bath, and then inoculate them into an α-MEM medium containing 10% FBS and double antibody. Culture them under the conditions of 5% O2, 5% CO2, and 37°C until the cell confluence reaches 70% to obtain a sufficient amount of human mesenchymal stem cells, which are used as the first mesenchymal stem cells.
[0038] S2. Induction culture: Place the first mesenchymal stem cells obtained in S1 into the induction medium described in Example 1 and culture them under the conditions of 5% O2, 5% CO2, and 37°C for 24 h to obtain the second mesenchymal stem cells.
[0039] S3. Harvest culture: Discard the culture solution obtained in S2, wash the cells twice with 10 mL of physiological saline, digest and harvest the cells with trypsin, and then inoculate them into an ultra-low attachment 96-well plate at an inoculation density of 1×10 6 cells / mL. Centrifuge at 300 g for 3 min, add 100 μL of α-MEM medium containing 10% human AB serum to each well, and culture them under the conditions of 5% O2, 5% CO2, and 37°C for 12 h.
[0040] Example 5: A method for increasing the IL-10 secretion of mesenchymal stem cells
[0041] S1. Amplification culture: Resuscitate human umbilical cord mesenchymal stem cells in a 37°C water bath, and then inoculate them into an α-MEM medium containing 10% FBS and double antibody. Culture them under the conditions of 5% O2, 5% CO2, and 37°C until the cell confluence reaches 70% to obtain a sufficient amount of human mesenchymal stem cells, which are used as the first mesenchymal stem cells.
[0042] S2. Inductive culture: Place the first mesenchymal stem cells obtained in S1 into the induction medium described in Example 2, and culture them under the conditions of 5% O2, 5% CO2, and 37°C for 24 h to obtain the second mesenchymal stem cells.
[0043] S3. Harvest culture: Discard the culture medium obtained in the culture of S2, wash the cells twice with 10 mL of normal saline, digest and harvest the cells with trypsin, then inoculate them into an ultra-low attachment 96-well plate at an inoculation density of 1×10 6 cells / mL, centrifuge at 300 g for 3 min, add 100 μL of α-MEM medium containing 10% human AB serum to each well, and culture them under the conditions of 5% O2, 5% CO2, and 37°C for 12 h.
[0044] Example 6: A method for increasing the secretion amount of IL-10 by mesenchymal stem cells
[0045] S1. Amplification culture: Resuscitate human umbilical cord mesenchymal stem cells in a 37°C water bath, then inoculate them into α-MEM medium containing 10% FBS and double antibodies, and culture them under the conditions of 5% O2, 5% CO2, and 37°C until the cell confluence reaches 70% to obtain a sufficient amount of human mesenchymal stem cells as the first mesenchymal stem cells.
[0046] S2. Inductive culture: Place the first mesenchymal stem cells obtained in S1 into the induction medium described in Example 3, and culture them under the conditions of 5% O2, 5% CO2, and 37°C for 24 h to obtain the second mesenchymal stem cells.
[0047] S3. Harvest culture: Discard the culture medium obtained in the culture of S2, wash the cells twice with 10 mL of normal saline, digest and harvest the cells with trypsin, then inoculate them into an ultra-low attachment 96-well plate at an inoculation density of 1×10 6 cells / mL, centrifuge at 300 g for 3 min, add 100 μL of α-MEM medium containing 10% human AB serum to each well, and culture them under the conditions of 5% O2, 5% CO2, and 37°C for 12 h.
[0048] To verify the secretion levels of IL-10 in human umbilical cord mesenchymal stem cells induced by the three culture media provided in Examples 1-3, the human umbilical cord mesenchymal stem cells obtained in Examples 4-6 were processed as follows in the present invention: Place the 96-well plate in an -80°C refrigerator and freeze for 8 hours, then thaw at room temperature, repeat 2 times, and collect the cell lysate; centrifuge the cell lysate at 3000g for 20 min to obtain the cell supernatant, then centrifuge at 10000g for 30 min to harvest the supernatant. Then, use the BCA method to detect the total protein concentration in the supernatant and use the ELISA method to detect the concentration of IL-10 in the supernatant. At the same time, human umbilical cord mesenchymal stem cells cultured under normoxic conditions without adding any cytokines, human umbilical cord mesenchymal stem cells cultured under hypoxic conditions without adding any cytokines, human umbilical cord mesenchymal stem cells cultured under normoxic conditions with IFN-γ and TNF-α (the addition amounts are the same as in Example 1), and human umbilical cord mesenchymal stem cells cultured under hypoxic conditions with IFN-γ and TNF-α (the addition amounts are the same as in Example 1) were used as controls. The detection results of the total protein concentration in the supernatant are shown in Figure 1 , and it can be seen from Figure 1 that the total protein concentration in the supernatant of human umbilical cord mesenchymal stem cells obtained in Examples 4-6 was significantly increased compared with various controls. The detection results of the concentration of IL-10 in the supernatant are shown in Figure 2 , and it can be seen from Figure 2 that the concentration of IL-10 in the supernatant of human umbilical cord mesenchymal stem cells obtained in Examples 4-6 was significantly increased compared with various controls. The above results indicate that the use of the induction culture medium and induction method provided by the present invention can significantly increase the IL-10 secretion level of mesenchymal stem cells.
[0049] To verify the effect of the induction culture medium provided by the present invention on the proliferation ability of human umbilical cord mesenchymal stem cells, the proliferation ability of human umbilical cord mesenchymal stem cells cultured with the induction culture medium described in Example 1 was detected. The specific method: Prepare a human umbilical cord mesenchymal stem cell suspension and count, inoculate 5×10 3 cells per well into a 96-well plate, add the induction culture medium described in Example 1, and the culture conditions are 37°C, 20% O2, 5% CO2. At the same time, use the basal medium added with IFN-γ and TNF-α (the addition amounts are the same as in Example 1) as the control group. On the 2nd, 4th, and 6th days after culturing, add 10 μL of CCK-8 to each well, gently mix, and measure the absorbance at 450 nm after incubating for 2 hours to establish a relationship curve between the culture time and the absorbance. It can be seen from Figure 3 that compared with the control group, the induction culture medium provided in Example 1 can significantly improve the proliferation ability of human umbilical cord mesenchymal stem cells.
[0050] Although the present invention has been disclosed above with the preferred embodiments, it is not intended to limit the present invention. Any person skilled in this technology can make various modifications and decorations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.
Claims
1. An induction medium for increasing the secretion amount of IL-10 by mesenchymal stem cells, characterized in that, The induction medium is obtained by adding cytokines to a basal medium. The cytokines are a human IFN-γ mimetic peptide and a TNF-α mimetic peptide. The amino acid sequence of the human IFN-γ mimetic peptide is as shown in SEQ ID NO.1, and the corresponding amino acid sequence of the TNF-α mimetic peptide is NKHNRKI.
2. The induction medium according to claim 1, wherein The addition amount of the human IFN-γ mimetic peptide is 200-1000 U / mL, and the addition amount of the TNF-α mimetic peptide is 5-10 ng / mL.
3. The induction medium according to claim 2, wherein The addition amount of the human IFN-γ mimetic peptide is 600 U / mL, and the addition amount of the TNF-α mimetic peptide is 10 ng / mL.
4. The induction medium according to claim 1, characterized in that, The basal medium is α-MEM medium containing double antibiotics and 10-20% FBS or double antibiotics and 10-20% human AB serum.
5. Use of the induction medium according to any one of claims 1-4 in increasing the secretion amount of IL-10 by mesenchymal stem cells.
6. A method for increasing the secretion amount of IL-10 by mesenchymal stem cells, characterized in that, Comprising the following steps: S1. Amplification culture: Amplify and culture to obtain a sufficient amount of human mesenchymal stem cells as the first mesenchymal stem cells. S2. Induction culture: Place the first mesenchymal stem cells obtained in S1 in the induction medium according to any one of claims 1-4, and culture at 2-5% O2, 5% CO2, and 37 °C for 12-24 h to obtain the second mesenchymal stem cells. S3. Harvest and culture: Discard the culture medium obtained in the culture described in S2, wash the cells twice with physiological saline, digest and harvest the cells with trypsin, and then inoculate them into an ultra-low attachment 96-well plate at an inoculation density of 1×10 6 cells / mL. Centrifuge the cells, add α-MEM medium containing 5-10% human AB serum, and culture them at 5% O2, 5% CO2, and 37°C for 12 h.
7. The method according to claim 6, wherein The amplification culture in S1 is to resuscitate human mesenchymal stem cells in a 37 °C water bath, and then inoculate them into the basal medium, and culture at 5% O2, 5% CO2, and 37 °C until the cell confluence reaches 70%-90%; the basal medium is α-MEM medium containing double antibiotics and 10-20% FBS or double antibiotics and 10-20% human AB serum.
8. The method according to claim 6, characterized in that The human mesenchymal stem cells in S1 are human bone marrow mesenchymal stem cells, human umbilical cord mesenchymal stem cells, or human placental chorionic mesenchymal stem cells.
Citation Information
Patent Citations
Active C-terminal peptides of interferon-gamma and their use
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