Application of Notch2 overexpression lentivirus in promoting aTreg cell differentiation

Abstract

The invention discloses an application of Notch2 overexpression lentivirus in promoting aTreg cell differentiation, and relates to the field of immune cell culture, and the application comprises the following steps: suspending PBMC in a T cell culture medium, laying a plate, adding Notch2 overexpression lentivirus, and culturing at 36.8-37.2 DEG C. The invention also discloses a preparation method of the Notch2 overexpression lentivirus. According to the application, the Notch2 overexpression lentivirus and the PBMC (peripheral blood mononuclear cell) are co-cultured on a culture medium, and the PBMC (peripheral blood mononuclear cell) is transfected by utilizing the Notch2 overexpression lentivirus, so that Notch2 protein molecules are overexpressed, and the number of Treg cells and aTreg cells in lymphocytes is increased.

Description

Technical Field

[0001] The present application relates to the field of immune cell culture, and particularly to the application of a lentivirus overexpressing Notch2 in promoting the differentiation of aTreg cells. 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 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, the present application provides the application of a lentivirus overexpressing Notch2 in promoting the differentiation of aTreg cells. In the present application, the lentivirus overexpressing Notch2 is co-cultured with PBMC (peripheral blood mononuclear cells) on a culture medium, and the PBMC (peripheral blood mononuclear cells) is transfected with the lentivirus overexpressing Notch2, so that the Notch2 protein molecule is overexpressed, and the numbers of Treg cells and aTreg cells in lymphocytes are increased.

[0005] The application of a lentivirus overexpressing Notch2 in promoting the differentiation of aTreg cells provided by the present application adopts the following technical solution:

[0006] The application of a lentivirus overexpressing Notch2 in promoting the differentiation of aTreg cells includes the following steps: suspending PBMC in a culture medium and then plating, and then adding the lentivirus overexpressing Notch2, and culturing under the temperature condition of 36.8 - 37.2 °C.

[0007] Preferably, the genome of the lentivirus overexpressing Notch2 includes a first nucleotide sequence for encoding a Notch2 receptor.

[0008] Preferably, the first nucleotide sequence is as shown in SEQ ID NO:1.

[0009] Preferably, the culture medium includes a basal cell culture medium, fetal bovine serum, antibiotics, L-glutamine, and a pH buffer.

[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 of penicillin and 80 - 120 μg / mL of streptomycin.

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

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

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

[0017] Preferably, the PBMC is prepared by the following method: taking peripheral blood, adding PBS buffer solution for dilution to obtain diluted blood, adding the diluted blood to lymphocyte separation solution, centrifuging at a rotation speed of 2300 - 2700 r / min for 15 - 25 min, sucking the monocyte layer, and adding PBS buffer solution for washing to obtain the PBMC.

[0018] Preferably, the PBMC is prepared by the following method: taking peripheral blood, adding PBS buffer solution for dilution to obtain diluted blood, adding the diluted blood to lymphocyte separation solution, centrifuging at a rotation speed of 2500 r / min for 20 min, sucking the monocyte layer, and adding PBS buffer solution for washing to obtain the PBMC.

[0019] Preferably, the culture includes semi - volume medium replacement after culturing for 15 h and continuing to culture for 48 h.

[0020] In summary, the present application includes at least one of the following beneficial technical effects:

[0021] 1. In the present application, by co - culturing the Notch2 - overexpressing lentivirus with PBMC (peripheral blood mononuclear cells) on T - cell medium, and transfecting PBMC (peripheral blood mononuclear cells) with the Notch2 - overexpressing lentivirus, the overexpression of Notch2 protein molecules is achieved, and the numbers of Treg cells and aTreg cells in lymphocytes are increased;

[0022] 2. After being treated with the Notch2 - overexpressing lentivirus in the present application, compared with 2.55% of Treg cells and 29.8% of aTreg cells in lymphocytes with low expression of Notch2 protein molecules, the Treg cells in lymphocytes reach 4.52% and the aTreg cells reach 77.1%;

[0023] 3. This application promotes the differentiation of aTreg cells, which is beneficial for promoting the differentiation of aTreg cells in patients lacking aTreg cells themselves and restoring the immune balance of the body. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a comparison chart of the protein blot detection results of the overexpressed lymphocytes of the Notch2 protein molecule in Example 1 and the lymphocytes in Comparative Examples 1-3;

[0025] Figure 2 It is a comparison chart of the flow cytometry detection results of the overexpressed lymphocytes of the Notch2 protein molecule in Example 1 and the lymphocytes in Comparative Examples 1-3. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0026] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the following further elaborates on the technical solutions of the present invention in combination with 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.

[0027] Example 1

[0028] The specific steps of the application of an overexpressed lentivirus of Notch2 in promoting the differentiation of aTreg cells provided in Example 1 of this application are as follows:

[0029] (1) PBMC extraction: Take 4 ml of peripheral blood from a patient 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) Virus transfection: Suspend PBMC in a culture medium (basic 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)), after resuspension, plate 1×106 PBMC in a 24-well plate, add the overexpressed lentivirus of Notch2 to transfect the cells, culture in an incubator at 37°C for 15 h, then perform half-volume medium replacement, and continue to culture for 48 hours to collect the overexpressed lymphocytes of the Notch2 protein molecule.

[0031] In this example, the inclusion criteria for patients with allergic rhinitis are as follows: 1. Patient symptoms: The presence of more than two symptoms such as clear nasal discharge, sneezing, nasal itching, nasal congestion, etc., with the symptoms lasting or accumulating for more than 1 hour per day, or accompanied by symptoms such as eye itching, tearing, and red eyes; 2. Physical signs: Common pale and edematous nasal mucosa, and clear nasal secretions in the nasal cavity; 3. Allergen basis: Positive for dust mite allergen in patients or combined with positive for one or more other allergens. 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] The overexpressed Notch2 lentivirus in this example was provided by Yuan Biotechnology Co., Ltd., and the nucleotide sequence of its genome is shown in SEQ ID NO:1, and the first nucleotide sequence for encoding the Notch2 receptor is shown in SEQ ID NO:2.

[0033] Comparative Example 1

[0034] Comparative Example 1 of this application provides a kind of lymphocyte, which is obtained by the following steps:

[0035] (1) Collect 4 ml of peripheral whole blood from healthy control patients, add 4 ml of PBS to the EP tube at a ratio of 1:1 and mix well to dilute the whole blood;

[0036] (2) Slowly add the diluted whole blood above 4 ml of lymphocyte separation solution;

[0037] (3) Centrifuge the EP tube in a centrifuge at a rotation speed of 2000 r / min at room temperature for 30 min. After centrifugation, use a pipette to aspirate the uppermost plasma layer, and collect the middle buffy coat layer, which is the lymphocyte;

[0038] (4) Add 3 volumes of PBS to the lymphocytes, centrifuge and discard the supernatant;

[0039] (5) Add 1 ml of RPMI1640 medium to the cell pellet to resuspend the cells, and obtain a lymphocyte suspension.

[0040] Comparative Example 2

[0041] Comparative Example 2 of this application provides a kind of lymphocyte, which is obtained by the following steps:

[0042] (1) Collect 4 ml of peripheral whole blood from patients with allergic rhinitis, add 4 ml of PBS to the EP tube at a ratio of 1:1 and mix well to dilute the whole blood;

[0043] (2) Slowly add the diluted whole blood above the 4 ml lymphocyte separation solution;

[0044] (3) Centrifuge the EP tube in a centrifuge at a rotational speed of 2000 r / min at room temperature for 30 min. After centrifugation, use a pipette to aspirate the uppermost plasma layer, and collect the middle buffy coat layer, which is the lymphocytes;

[0045] (4) Add 3 volumes of PBS to the lymphocytes, and centrifuge to discard the supernatant;

[0046] (5) Add 1 ml of RPMI1640 medium to the cell pellet, and resuspend the cells to obtain a lymphocyte suspension.

[0047] Comparative Example 3

[0048] Comparative Example 3 of the present application provides a kind of lymphocytes, which are obtained by the following steps: (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 on the lymphocyte separation solution (TBD, LTS1077), centrifuge at a rotational speed of 2500 r / min for 20 minutes, aspirate the middle monocyte layer, and obtain PBMC after washing with PBS buffer.

[0049] (2) Virus transfection: Suspend the PBMC in complete T cell medium (1640, 10% FBS, 100 U / Ml penicillin, 100 μg / mL streptomycin, 5 μmol / L L-glutamine and 1×HEPES), after resuspension, plate 1×106 PBMC in a 24-well plate, add lentivirus overexpressing Notch2 to transfect the cells, and add 10 μmol / L Notch2 inhibitor, culture in an incubator at 37 °C for 15 h and then perform half-volume medium change, continue to culture for 48 hours, and collect the lymphocytes.

[0050] Test detection

[0051] (1) The overexpressed lymphocytes of Notch2 protein molecule obtained from the application of Notch2 overexpressing lentivirus of Example 1 in promoting the differentiation of aTreg cells, the lymphocytes of Comparative Example 1, the lymphocytes of Comparative Example 2, and the lymphocytes of Comparative Example 3 were respectively detected for the expression of Notch2. The specific steps are as follows: After centrifuging the cells, the supernatant was discarded, and RIPA lysis buffer (containing phosphatase inhibitor and protease inhibitor 2 mg / ml) was added. After the cell lysis was completed, 4× protein loading buffer was added, and it was boiled at 95 °C for 10 minutes. The extracted protein was electrophoretically separated in an SDS-PAGE gel, and then the electrophoretically separated protein was transferred onto a PVDF membrane (GE Life, USA, #10600023). After the membrane transfer was completed, it was placed in 5% skim milk for blocking for 1 hour. The blocked membrane was washed 3 times with TBS-Tween20, and then sequentially placed in the corresponding primary antibodies Notch2 (Sigma, 07-1234) and GAPDH (Servicebio, GB15002-100) and incubated overnight at 4 °C. The concentrations of these antibodies were prepared according to the instructions. The next day, the incubated membrane was washed 3 times with TBS-Tween20. After the washing was completed, it was placed in a chemical secondary antibody (1:20000) and incubated at room temperature for 1 hour. After the incubation was completed, it was washed 3 times with TBS-Tween20 again. The washed membrane was placed in ECL luminescent solution for activation, and the activated membrane was placed in an imager for development, and the expression results were obtained as Figure 1 shown, where the result of Example 1 was marked as overexpressed Notch2, the result of Comparative Example 1 was marked as the control group, the result of Comparative Example 2 was marked as low-expressed Notch2, and the result of Comparative Example 3 was marked as overexpressed Notch2 + inhibitor.

[0052] (2) The overexpressed lymphocytes of Notch2 protein molecule obtained from the application of Notch2 overexpressing lentivirus of Example 1 in promoting the differentiation of aTreg cells, the lymphocytes of Comparative Example 1, the lymphocytes of Comparative Example 2, and the lymphocytes of Comparative Example 3 were respectively detected for the number of Treg cells and the number of aTreg cells. The specific steps are as follows: The cells were resuspended and washed twice with PBS buffer, centrifuged at 370 g for 6 minutes, and surface staining antibodies CD4-BV421 (BD, 562424), CD25-FITC (BD, 564467), CD127-APC (BD, 558598), and CD45RA (BD, 558489) were added. It was incubated in the dark at 4 °C for 30 minutes. After the incubation was completed, it was centrifuged at 370 g for 6 minutes, the supernatant was discarded and PBS buffer was added for washing. After washing 2 times, it was detected for the number of Treg cells on a Beckman flow cytometer as Figure 2 (a) shown, and the number of aTreg cells as Figure 2As shown in (b). Among them, the results of Example 1 are marked as Notch2 overexpression, the results of Comparative Example 1 are marked as the control group, the results of Comparative Example 2 are marked as Notch2 low expression, and the results of Comparative Example 3 are marked as Notch2 overexpression + inhibitor.

[0053] Result analysis

[0054] The following Figure 1-2 Combined with the provided experimental results, the present application will be described in detail.

[0055] Referring to Figure 1 , it can be seen that the lymphocytes obtained by applying the Notch2 overexpressing lentivirus of Example 1 in promoting the differentiation of aTreg cells are lymphocytes with overexpression of Notch2 protein molecules. Comparative Example 1 is lymphocytes with normal expression of Notch2 protein molecules, Comparative Example 2 is lymphocytes with low expression of Notch2 protein molecules, and the expression level of Notch2 protein molecules in the lymphocytes of Comparative Example 3 is between the expression levels of Notch2 protein molecules in the lymphocytes of Comparative Example 1 and Example 1.

[0056] Referring to Figure 2 , it can be seen that the proportion of Treg cells in the lymphocytes obtained by applying the Notch2 overexpressing lentivirus of Example 1 in promoting the differentiation of aTreg cells is higher than the proportion of Treg cells in the lymphocytes of Comparative Example 2 and Comparative Example 3, reaching a level close to the proportion of Treg cells in the lymphocytes of Comparative Example 1. The proportion of aTreg cells in the Treg cells obtained by applying the Notch2 overexpressing lentivirus of Example 1 in promoting the differentiation of aTreg cells is higher than the proportion of aTreg cells in the Treg cells of Comparative Example 2 and Comparative Example 3, indicating that the application of the Notch2 overexpressing lentivirus of Example 1 of the present application in promoting the differentiation of aTreg cells is not only beneficial to increasing the proportion of Treg cells in lymphocytes, but also can increase the proportion of aTreg cells in Treg cells, which is beneficial to promoting the differentiation of aTreg cells.

[0057] This specific embodiment is only an interpretation of the present application and does not limit the present application. Those skilled in the art can make modifications without creative contributions to this embodiment according to needs after reading this specification, 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. Use of a Notch2 overexpressing lentivirus in promoting aTreg cell differentiation, characterized in that: The following steps are involved: PBMCs were suspended in culture medium and plated, and Notch2 overexpression lentivirus was added and cultured at 36.8-37.2°C.

2. The use of the Notch2 overexpressing lentivirus according to claim 1 in promoting aTreg cell differentiation, characterized in that: The genome of the Notch2 overexpression lentivirus includes a first nucleotide sequence for encoding a Notch2 receptor.

3. The use of the Notch2 overexpressing lentivirus according to claim 1 in promoting aTreg cell differentiation, characterized in that: The first nucleotide sequence is shown as SEQ ID NO:

2.

4. The use of the Notch2 overexpressing lentivirus in promoting aTreg cell differentiation according to claim 1, characterized in that: The culture medium includes basal cell culture medium, fetal bovine serum, antibiotics, L-glutamine and pH buffer.

5. The use of the Notch2 overexpressing lentivirus in promoting aTreg cell differentiation according to claim 4, characterized in that: The volume concentration of the fetal bovine serum is 8-12%.

6. The use of the Notch2 overexpressing lentivirus in promoting aTreg cell differentiation according to claim 4, characterized in that: The antibiotics include one or both of penicillin and streptomycin.

7. The use of the Notch2 overexpressing lentivirus in promoting aTreg cell differentiation according to claim 6, characterized in that: The antibiotics include 80-120 U / mL penicillin and 80-120 μg / mL streptomycin.

8. The use of Notch2 overexpressing lentivirus in promoting aTreg cell differentiation according to claim 4, characterized in that: The final concentration of L-glutamine was 4-6 μM.

9. The use of the Notch2 overexpressing lentivirus in promoting aTreg cell differentiation according to claim 1, characterized in that: The PBMC is prepared by the following method: taking peripheral blood and adding PBS buffer to dilute it to obtain diluted blood, adding the diluted blood to lymphocyte separation fluid, centrifuging at a speed of 2300-2700r / min for 15-25min, aspirating the mononuclear cell layer, adding PBS buffer to wash, and obtaining the PBMC.

10. The use of the Notch2 overexpressing lentivirus in promoting aTreg cell differentiation according to claim 1, characterized in that: The culturing includes replacing half of the medium after culturing for 13-17 hours, and continuing culturing for 45-50 hours.

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