Pharmaceutical composition for promoting aTreg cell differentiation and application thereof

Abstract

The invention discloses a pharmaceutical composition for promoting aTreg cell differentiation and application thereof, and relates to the field of biological medicine, and the pharmaceutical composition comprises zinc salt, a basic cell culture medium, fetal calf serum, antibiotic, L-glutamine and a pH buffer agent. According to the application, the basic cell culture medium is used for providing basic nutrition required by cell growth for lymphocytes, the fetal calf serum is used for providing growth factors, hormones, adhesion proteins and nutrient substances required by cell growth for the lymphocytes, and the antibiotics are used for preventing bacterial infection; l-glutamine provides key amino acid for cell energy metabolism and participates in synthesis of protein and nucleic acid, zinc salt provides zinc ions, and under the comprehensive action, the proportion of Treg cells and aTreg cells differentiated from lymphocytes in peripheral blood mononuclear cells is increased.

Description

Technical Field

[0001] This application relates to the field of immune cell culture, and particularly to a pharmaceutical composition for promoting the differentiation of aTreg cells and its application. Background Art

[0002] aTreg cells (activated Treg) are a subtype of regulatory T cells (Treg cells) - activated-like Treg cells. They have a strong immunosuppressive function, the ability to migrate into tissues, play an important inhibitory role in the inflammatory response in tissues, and play an important role in suppressing immune activity functions and in autoimmune diseases.

[0003] aTreg cells are differentiated from immune cells. Therefore, how to promote the differentiation of immune cells into aTreg cells has important scientific research and clinical application values. Summary of the Invention

[0004] In view of the deficiencies of the above related technologies, this application provides a pharmaceutical composition for promoting the differentiation of aTreg cells and its application. In this application, the basic cell culture medium provides the basic nutrients required for cell growth for lymphocytes, fetal bovine serum provides growth factors, hormones, adhesion proteins and nutrients required for cell growth for lymphocytes, antibiotics prevent bacterial infections, L-glutamine provides key amino acids for cell energy metabolism and participates in the synthesis of proteins and nucleic acids, and zinc salts provide zinc ions. Under the combined action, the proportion of Treg cells differentiated from lymphocytes in peripheral blood mononuclear cells and the proportion of aTreg cells in Treg cells are increased.

[0005] On the one hand, a pharmaceutical composition for promoting the differentiation of aTreg cells provided by this application adopts the following technical solution: A pharmaceutical composition for promoting the differentiation of aTreg cells, comprising a zinc salt, a basic cell culture medium, fetal bovine serum, an antibiotic, L-glutamine and a pH buffer.

[0006] Preferably, the final concentration of the zinc salt is 25 - 150 μM.

[0007] Preferably, the final concentration of the zinc salt is 100 μM.

[0008] Preferably, the zinc salt includes one or more of zinc sulfate, zinc chloride, and zinc gluconate.

[0009] Preferably, the zinc salt is zinc sulfate.

[0010] Preferably, the volume concentration of the fetal bovine serum is 8 - 12%.

[0011] Preferably, the volume concentration of the fetal bovine serum is 10%.

[0012] Preferably, the antibiotic includes one or both of penicillin and streptomycin.

[0013] Preferably, the antibiotic includes 80 - 120 U / mL penicillin and 80 - 120 μg / mL streptomycin.

[0014] Preferably, the antibiotic includes 100 U / mL penicillin and 100 μg / mL streptomycin.

[0015] Preferably, the final concentration of L - glutamine is 4 - 6 μM.

[0016] Preferably, the final concentration of L - glutamine is 5 μM.

[0017] On the other hand, the application of a pharmaceutical composition for promoting the differentiation of aTreg cells provided by the present application adopts the following technical solution: The application of the above - mentioned pharmaceutical composition in promoting the differentiation of aTreg cells.

[0018] Preferably, the application steps are as follows: peripheral blood mononuclear cells are placed in the pharmaceutical composition and cultured for 10 - 14 h.

[0019] The application of the above - mentioned pharmaceutical composition in promoting the differentiation of Treg cells.

[0020] In summary, the present application includes at least one of the following beneficial technical effects: 1. The present application provides basic nutrients required for cell growth for lymphocytes through a basic cell culture medium, growth factors, hormones, adhesion proteins, and nutrients required for cell growth for lymphocytes through fetal bovine serum, antibiotics prevent bacterial infection, L - glutamine provides key amino acids for cell energy metabolism and participates in the synthesis of proteins and nucleic acids, and zinc salts provide zinc ions. Under the combined action, the proportion of Treg cells differentiated from lymphocytes in peripheral blood mononuclear cells is increased; 2. The present application provides basic nutrients required for cell growth for lymphocytes through a basic cell culture medium, growth factors, hormones, adhesion proteins, and nutrients required for cell growth for lymphocytes through fetal bovine serum, antibiotics prevent bacterial infection, L - glutamine provides key amino acids for cell energy metabolism and participates in the synthesis of proteins and nucleic acids, and zinc salts provide zinc ions. Under the combined action, the proportion of aTreg cells differentiated from Treg cells in lymphocytes in peripheral blood mononuclear cells is increased; 3. The pharmaceutical composition of the present application is beneficial to increasing the proportion of Treg cell differentiation and at the same time increasing the proportion of aTreg cell differentiation. The two effects produce a synergistic effect and further increase the total amount of aTreg cells. Description of the Drawings

[0021] Figure 1 It is a comparison chart of the proportion of Treg cells in the flow cytometry detection results of lymphocytes in Application Examples 1-4 and Application Comparative Example 1.

[0022] Figure 2 It is a comparison chart of the proportion of aTreg cells in the flow cytometry detection results of lymphocytes in Application Examples 1-4 and Application Comparative Example 1. Detailed implementation manners

[0023] In order to make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention will be further described in detail below in conjunction with the embodiments. The equipment and reagents used in each embodiment and test example can be obtained from commercial channels without special instructions. The specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0024] Embodiment 1 Embodiment 1 of the present application provides a pharmaceutical composition for promoting the differentiation of aTreg cells, which is composed of the following components: basal cell culture medium (RPMI 1640), fetal bovine serum (volume fraction 10%), penicillin (100 U / mL), streptomycin (100 μg / mL), L-glutamine (5 μM), zinc sulfate (25 μM), and pH buffer (1×HEPES).

[0025] Embodiment 2 Embodiment 2 of the present application provides a pharmaceutical composition for promoting the differentiation of aTreg cells. The difference between Embodiment 2 and Embodiment 1 is that: the final concentration of zinc sulfate in Embodiment 2 is 50 μM.

[0026] Embodiment 3 Embodiment 3 of the present application provides a pharmaceutical composition for promoting the differentiation of aTreg cells. The difference between Embodiment 3 and Embodiment 1 is that: the final concentration of zinc sulfate in Embodiment 3 is 100 μM.

[0027] Embodiment 4 Embodiment 4 of the present application provides a pharmaceutical composition for promoting the differentiation of aTreg cells. The difference between Embodiment 4 and Embodiment 1 is that: the final concentration of zinc sulfate in Embodiment 4 is 150 μM.

[0028] Comparative Example 1 Comparative Example 1 provides a pharmaceutical composition. The difference between Comparative Example 1 and Embodiment 1 is that: zinc sulfate is not added in Comparative Example 1.

[0029] Application Example 1 Application Example 1 of the present application provides an application of a pharmaceutical composition for promoting the differentiation of aTreg cells in promoting the differentiation of aTreg cells. The application steps are as follows: (1)PBMC extraction: Take 4 ml of peripheral blood from patients with allergic rhinitis, add 4 ml of PBS buffer to dilute the blood, slowly add the diluted blood onto lymphocyte separation medium (TBD, LTS1077), centrifuge at a rotation speed of 2500 r / min for 20 minutes, aspirate the middle monocyte layer, and wash with PBS buffer to obtain PBMC.

[0030] (2)Culture: Suspend PBMC in the pharmaceutical composition of Example 1, after resuspension, plate 1×106 PBMC in a 24-well plate, change the medium after incubation for 15 min, continue to culture for 12 h, and collect the cells.

[0031] In this example, the inclusion criteria for patients with allergic rhinitis are as follows: 1. Patient symptoms: More than two of the symptoms such as runny nose with clear water-like discharge, sneezing, nasal itching, nasal congestion, etc., with the daily symptoms lasting or accumulating for more than 1 h, or accompanied by symptoms such as eye itching, tearing, and red eyes; 2. Signs: Common pale and edematous nasal mucosa, clear water-like nasal secretions; 3. Allergen basis: The patient is positive for dust mite allergen or combined with one or more other allergens being positive. All patients have no history of systemic diseases, malignant tumors, etc. And these patients have not been treated with immunosuppressive drugs such as glucocorticoids. Approved by the Ethics Committee of Renmin Hospital of Wuhan University (Ethical Approval Number: WDRY2023-K143), and each patient signed a written informed consent form. Peripheral blood was drawn after signing the informed consent form.

[0032] Application Example 2 Application Example 2 of the present application provides an application of a pharmaceutical composition for promoting the differentiation of aTreg cells in promoting the differentiation of aTreg cells. The difference between Application Example 2 and Application Example 1 is that in Application Example 2, the pharmaceutical composition of Example 2 is used to replace the pharmaceutical composition of Example 1.

[0033] Application Example 3 Application Example 3 of the present application provides an application of a pharmaceutical composition for promoting the differentiation of aTreg cells in promoting the differentiation of aTreg cells. The difference between Application Example 3 and Application Example 1 is that in Application Example 3, the pharmaceutical composition of Example 3 is used to replace the pharmaceutical composition of Example 1.

[0034] Application Example 4 Application Example 4 of the present application provides an application of a pharmaceutical composition for promoting the differentiation of aTreg cells in promoting the differentiation of aTreg cells. The difference between Application Example 4 and Application Example 1 is that in Application Example 4, the pharmaceutical composition of Example 4 is used to replace the pharmaceutical composition of Example 1.

[0035] Application Comparative Example 1 Application Comparative Example 1 provides an application of a pharmaceutical composition. The difference between Application Comparative Example 1 and Application Example 1 is that in Application Comparative Example 1, the pharmaceutical composition of Comparative Example 1 is used to replace the pharmaceutical composition of Example 1.

[0036] Test Detection (1) Detect the number of Treg cells and the number of aTreg cells in the finally collected cells of Application Examples 1-4 and Application Comparative Example 1. The specific steps are as follows: Resuspend and wash the cells twice with PBS buffer, centrifuge at 370 g for 6 minutes, and add surface staining antibodies CD4-BV421 (BD, 562424), CD25-FITC (BD, 564467), CD127-APC (BD, 558598), CD45RA (BD, 558489). Incubate in the dark at 4 °C for 30 minutes. After incubation, centrifuge at 370 g for 6 minutes, pour out the supernatant and add PBS buffer for washing. After washing 2 times, detect the proportion of Treg cells on a Beckman flow cytometer as Figure 1 shown, and the proportion of aTreg cells is as Figure 2 shown. Among them, the result of Application Example 1 is marked as 25 μM, the result of Application Example 2 is marked as 50 μM, the result of Application Example 3 is marked as 100 μM, the result of Application Example 4 is marked as 150 μM, and the result of Application Comparative Example 1 is marked as Control.

[0037] Result Analysis The following combines Figure 1-2 the provided experimental results to elaborate on this application in detail.

[0038] Referring to Figure 1 , it can be seen that the proportion of Treg cells in the lymphocytes obtained from Application Examples 1-4 using the pharmaceutical compositions for promoting aTreg cell differentiation in Examples 1-4 is higher than the proportion of Treg cells in Application Comparative Example 1 using the pharmaceutical composition of Comparative Example 1. Referring to Figure 2It can be seen that the proportion of aTreg cells in the lymphocytes obtained from Application Examples 1-4 of the pharmaceutical composition for promoting aTreg cell differentiation in Examples 1-4 is higher than that in Application Comparative Example 1 of the pharmaceutical composition in Comparative Example 1. This shows that after the pharmaceutical composition for promoting aTreg cell differentiation in Examples 1-4 of the present application is used to treat peripheral blood mononuclear cells, it is not only beneficial to increase the proportion of Treg cells in lymphocytes, but also can increase the proportion of aTreg cells in Treg cells, which is conducive to promoting aTreg cell differentiation. Moreover, the proportions of Treg cells (10.8%) and aTreg cells (52.9%) in the lymphocytes obtained from Application Example 3 of the pharmaceutical composition for promoting aTreg cell differentiation in Example 3 are higher than those of Treg cells and aTreg cells in Application Comparative Example 1, Application Example 1, Application Example 2, and Application Example 4 of the pharmaceutical compositions in Comparative Example 1, Example 1, Example 2, and Example 4. This shows that controlling the final concentration of zinc sulfate in the pharmaceutical composition to be 100 μM is beneficial to promoting the differentiation of Treg cells and aTreg cells.

[0039] This specific embodiment is only an interpretation of the present application and does not limit the present application. After reading this specification, those skilled in the art can make modifications to this embodiment without creative contributions as needed, but as long as they are within the scope of the claims of the present application, they are protected by the patent law.

Claims

1. A pharmaceutical composition for promoting the differentiation of aTreg cells, characterized in that: It includes zinc salts, basal cell culture medium, fetal bovine serum, antibiotics, L-glutamine and pH buffer.

2. The pharmaceutical composition for promoting the differentiation of aTreg cells according to claim 1, wherein: The final concentration of the zinc salts is 25 - 150 μM.

3. The pharmaceutical composition for promoting the differentiation of aTreg cells according to claim 1, characterized in that: The zinc salts include one or more of zinc sulfate, zinc chloride, zinc gluconate.

4. The pharmaceutical composition for promoting the differentiation of aTreg cells according to claim 1, characterized in that: The volume concentration of the fetal bovine serum is 8 - 12%.

5. The pharmaceutical composition for promoting the differentiation of aTreg cells according to claim 1, wherein: The antibiotics include one or both of penicillin and streptomycin.

6. The pharmaceutical composition for promoting the differentiation of aTreg cells according to claim 5, wherein: The antibiotics include 80 - 120 U / mL penicillin and 80 - 120 μg / mL streptomycin.

7. The pharmaceutical composition for promoting the differentiation of aTreg cells according to claim 1, wherein: The final concentration of the L-glutamine is 4 - 6 μM.

8. Use of the pharmaceutical composition according to any one of claims 1 - 7 in promoting the differentiation of aTreg cells.

9. The application according to claim 8, characterized in that: The application steps are as follows: Peripheral blood mononuclear cells are cultured in the pharmaceutical composition for 10 - 14 h.

10. Use of the pharmaceutical composition according to any one of claims 1 - 7 in promoting the differentiation of Treg cells.

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