A γδT cell preparation and its application

Abstract

The present invention provides a γδT cell preparation and its application, the γδT cell preparation includes γδT cell fluid, 100‑800μmol / L ergonovine maleate, 100‑700μmol / L moringin and / or 100‑1000μmol / L bergenin. The results of the study show that ergonovine maleate, moringin, bergenin and γδT cell combination are used to kill tumor cells, with good inhibitory activity, especially for lung cancer cells and thyroid cancer cells. The inhibition rate reaches about 60% and 90% respectively. The product formula of the present invention is clear and simple, easy to use, and conducive to promotion and application.

Description

Technical Field

[0001] The present invention relates to the technical field of cell preparation preparation, and in particular to a γδT cell preparation and application thereof. Background Art

[0002] According to the difference in surface antigen receptors (TCR), T cells can be divided into αβT cells and γδT cells. γδT cells recognize antigens through the γδTCR complex. Their cell subtypes, distribution locations and host immunity are different from those of αβT cells. Studies have found that γδT cells are closely related to tumor immunity. γδT cells are mainly found in barrier tissues such as skin and mucous membranes, and only account for CD3 + A small subset of T cells can enter an activated state within minutes of antigen stimulation. Because they can rapidly produce a variety of cytokines upon activation, γδT cells contribute to the first line of defense against infection and tumors. In addition to their inherent immune characteristics, γδT cells also possess adaptive immune functions. Human γδT cells can recognize melanoma-associated antigenic peptides through the MHC pathway. However, unlike αβT cells, not only antigens presented by MHC molecules can activate γδT cells through the γδTCR. For example, phosphoantigens (PAgs) expressed by tumors and various cytokines can act on the γδTCR to activate cells.

[0003] The inherent and adaptive immune properties of γδT cells make them a key immunotherapy cell type. They offer enhanced safety and minimal off-target effects for tumor immunotherapy. Furthermore, because antigen recognition is not restricted by the MHC, γδT cells can be isolated and expanded from healthy individuals. They can also be combined with CAR-T therapy to generate sufficient cell subsets, reducing byproducts during expansion and lowering treatment costs. γδT cells are capable of killing not only autologous tumor cells but also allogeneic tumor cells without toxicity to normal cells. Currently, γδT cells have been reported to exhibit anti-tumor activity against breast cancer, kidney cancer, pancreatic cancer, colon cancer, melanoma, and other tumors.

[0004] Because γδT cells possess potent direct cytotoxicity against tumor cells and can synergize with other immune cells to exert their anti-tumor activity, they have become a new research hotspot in cellular immunotherapy. As γδT cell immunotherapy has gained attention and become a current research hotspot, related cell preparations have also garnered attention. Current γδT cell preparations primarily consist of γδT cell fluids, which, while effective in treating tumors, have limited activity against tumor cells and are insufficient to meet patient needs. Summary of the Invention

[0005] In view of the deficiencies of the prior art, the present invention aims to provide a γδT cell preparation and its application.

[0006] The technical solutions of the present invention are as follows:

[0007] A γδT cell preparation containing γδT cell fluid and at least one of 100-600 μmol / L ergonovine maleate, 100-700 μmol / L moringin, and 100-1000 μmol / L bergenin. It is recommended to mix the preparation immediately before use.

[0008] Furthermore, the cell concentration in the γδT cell solution is 2×10 7 ~10×10 7 The cell concentration was adjusted with RPMI-1640 medium.

[0009] Furthermore, the γδT cell preparation contains γδT cell fluid, and at least one of 600 μmol / L ergonovine maleate, 600 μmol / L moringin, and 600 μmol / L bergenin.

[0010] Furthermore, the γδT cell preparation contains γδT cell fluid, 200 μmol / L ergonovine maleate, 200 μmol / L moringin and 200 μmol / L bergenin.

[0011] On the other hand, the present invention also provides the use of the γδT cell preparation in the preparation of anti-tumor drugs.

[0012] Furthermore, the tumor includes lung cancer and thyroid cancer.

[0013] Compared with the prior art, the present invention has the following advantages:

[0014] The product of the present invention includes γδT cell fluid, ergometrine maleate, moringin, and bergenin. Research results show that the combination of ergometrine maleate, moringin, and bergenin with γδT cells has good inhibitory activity against tumor cells, especially against lung cancer cells and thyroid cancer cells, with inhibition rates reaching approximately 60% and 90%, respectively.

[0015] The product of the present invention has a clear and simple formula, is easy to use, and is conducive to promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 : The killing rate of each group of cell preparations on tumor cells. DETAILED DESCRIPTION

[0017] In order to enable those skilled in the art to better understand the technical content of the present invention, the present invention is further described below in conjunction with specific embodiments and drawings.

[0018] The γδT cells described in the following examples were derived from healthy human peripheral blood and isolated according to conventional methods. 23 H 27 N3O6), moringin (CAS No. 73255-40-0), and bergenin (CAS No. 477-90-7) are all commercially available products.

[0019] Example 1

[0020] A γδT cell preparation containing γδT cell fluid (2×10 7 ~10×10 7 / ml), 600μmol / L ergometrine maleate, 600μmol / L moringin and 600μmol / L bergenin.

[0021] Example 2

[0022] A γδT cell preparation containing γδT cell fluid (2×10 7 ~10×10 7 / ml), 600μmol / L ergonovine maleate.

[0023] Example 3

[0024] A γδT cell preparation containing γδT cell fluid (2×10 7 ~10×10 7 / ml), 600μmol / L Moringa seed extract.

[0025] Example 4

[0026] A γδT cell preparation containing γδT cell fluid (2×10 7 ~10×10 7 / ml), 600μmol / L bergenin.

[0027] Example 5

[0028] A γδT cell preparation containing γδT cell fluid (2×10 7 ~10×10 7 / ml), 200μmol / L ergometrine maleate, 200μmol / L moringin and 200μmol / L bergenin.

[0029] Example 6

[0030] A γδT cell preparation containing γδT cell fluid (2×10 7 ~10×10 7 / ml), 100μmol / L ergonovine maleate, 100μmol / L moringa seed extract.

[0031] Example 7

[0032] A γδT cell preparation containing γδT cell fluid (2×10 7 ~10×10 7 / ml), 700μmol / L moringin, 100μmol / L bergenin.

[0033] Example 8

[0034] A γδT cell preparation containing γδT cell fluid (2×10 7 ~10×10 7 / ml), 600μmol / L ergonovine maleate, and 100μmol / L bergenin.

[0035] Example 9

[0036] A γδT cell preparation containing γδT cell fluid (2×10 7 ~10×10 7 / ml), 500μmol / L ergometrine maleate, 500μmol / L moringin, 1000μmol / L bergenin.

[0037] Example 10

[0038] A γδT cell preparation containing γδT cell fluid (2×10 7 ~10×10 7 / ml), 500μmol / L ergometrine maleate, 500μmol / L moringin, 500μmol / L bergenin.

[0039] γδT cell preparation killing experiment

[0040] Lung cancer A549 cells (1×10 6 / mL), thyroid cancer cells SW579 (1×10 6 / mL) were inoculated into 96-well cell culture plates as target cells, 100 μl / well. The γδT cell preparation was adjusted to a cell count of 2×10 7 / ml, and added into the culture plate inoculated with target cells according to the following design, 100 μl / well, 6 replicate wells in each group, and repeated the experiment three times.

[0041] Experimental group 1: 100 μl target cells + 100 μl γδT cell preparation (2×10 7 / mlγδT cells)

[0042] Experimental group 2: 100 μl target cells + 100 μl γδT cell preparation (2×10 7 / mlγδT cells + 600μmol / L ergonovine maleate)

[0043] Experimental group 3: 100 μl target cells + 100 μl γδT cell preparation (2×10 7 / mlγδT cells + 600μmol / L Moringa seed extract)

[0044] Experimental group 4: 100 μl target cells + 100 μl γδT cell preparation (2×10 7 / mlγδT cells + 600μmol / L bergenin)

[0045] Experimental group 5: 100 μl target cells + 100 μl γδT cell preparation (2×10 7 / mlγδT cells + 200μmol / L ergonovine maleate + 200μmol / L moringin + 200μmol / L bergenin)

[0046] Target cell group: 100 μl target cells

[0047] Negative control group: 100 μl γδT cell preparation (2×10 7 / mlγδT cells)

[0048] Cells in each group were cultured in a 37°C, 5% CO2 incubator. After 24 hours, 20 μl / well of MTT solution (5 mg / ml) was added. Incubation was stopped 4 hours later, the supernatant was carefully aspirated, and 150 μl / well of dimethyl sulfoxide was added. The cells were gently mixed, and the OD values at a wavelength of 470 nm were measured using a microplate reader. The killing rate of each group was calculated. Analysis of variance was performed using SPSS software.

[0049] Killing rate = (OD value of target cell group + OD value of negative control group - OD value of experimental group) / OD value of target cell group × 100%

[0050] Experimental results:

[0051] The experimental results are shown in the table below. The results show that the killing rates of experimental groups 2 to 5 against thyroid cancer cells were significantly higher than those of experimental group 1, indicating that ergometrine maleate, moringa seed oil, and bergenin have a killing effect on thyroid cancer cells, or that ergometrine maleate, moringa seed oil, and bergenin can enhance the killing activity of γδT cells and thus enhance the killing effect of γδT cells on tumor cells.

[0052] The killing rates of experimental groups 3 to 5 against lung cancer cells were significantly or extremely significantly higher than those in experimental group 1, indicating that moringa seed element and bergenin have a killing effect on thyroid cancer cells, or that moringa seed element and bergenin can enhance the killing activity of γδT cells and thus enhance the killing effect of γδT cells on tumor cells.

[0053] In addition, by comparing Group 5 with other groups, it can be seen that the combination of ergonovine maleate, moringa seed extract, and bergenin exhibited more significant tumor killing activity when applied to γδT cell preparations.

[0054] Results of variance analysis of kill rate

[0055]

[0056] Post hoc multiple comparison results

[0057]

[0058] The above descriptions are only some embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A γδT cell preparation comprising a γδT cell fluid, characterized in that: It also contains 100-600 μmol / L ergometrine maleate, 100-700 μmol / L moringin, and 100-1000 μmol / L bergenin.

2. The γδT cell preparation according to claim 1, characterized in that The cell concentration in the γδT cell solution was 2×10 7 ~10×10 7 pcs / ml.

3. The γδT cell preparation according to claim 1, characterized in that Contains 600 μmol / L ergometrine maleate, 600 μmol / L moringin, and 600 μmol / L bergenin.

4. The γδT cell preparation according to claim 1, characterized in that Contains γδT cell fluid, 200 μmol / L ergonovine maleate, 200 μmol / L moringin, and 200 μmol / L bergenin.

5. Use of the γδT cell preparation according to any one of claims 1 to 4 in the preparation of an anti-tumor drug.

6. The use according to claim 5, characterized in that The tumors include lung cancer and thyroid cancer.

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